Transcription

1 Transfection Products

2 ONE FOCUS: TRANSFECTION Transfection is a type of macromolecular delivery in which large, negatively charged nucleic acids are delivered through cellular membranes and into the intracellular compartments where they can exert their function. Almost two decades after its founding, transfection continues to be the focal point and passion of Mirus Bio. Every transfection product offered by Mirus Bio is the result of scientific discovery and development from an in-house team of organic chemists, molecular biologists and cell biologists. When you contact Mirus Bio, you are speaking with a scientist with true expertise in the development, manufacture, and performance of the product. MOST RECENT TRANSFECTION BREAKTHROUGHS 13: TransIT-X2 Dynamic Delivery System For superior delivery of plasmid DNA and sirna TransIT -BrCa Transfection Reagent The first breast cancer cell transfection reagent 11: TransIT -3D Transfection Reagent For use in 3D cell culture scaffolds 1: TransIT-PRO Transfection Kit Large-scale, high yield protein production 9: TransIT - Transfection Reagent High performance broad spectrum transfection 8: Ingenio Electroporation Kits & Solution Versatile, multi-platform electroporation solution OUR PROMISE TO YOU: SERVICE THROUGH EXPERTISE Order Online 24/7 (Credit Cards or Purchase Orders) Technical and Customer Service Hours of Operation: 8: AM 4: PM Central Time, Monday Friday Technical Support techsupport@mirusbio.com Customer Service sales@mirusbio.com Phone: (toll free in the U.S.) or Fax: U.S. Distribution Direct: Fisher Scientific: International Distribution For a complete list of our worldwide distributors, please visit: and contact the distributor in your region. Mail Mirus Bio LLC Attention: Customer Service 545 Science Drive Madison, WI USA Request FREE Samples Start with our Reagent Agent transfection database to determine the best solution for your transfection needs: Two ways to request a sample: 1. Visit us at 2. Call us at

3 CONTENTS DNA/RNA TRANSFECTION BROAD SPECTRUM DNA & sirna/mirna NEW! TransIT-X2 Dynamic Delivery System DNA TRANSFECTION BROAD SPECTRUM DNA TransIT - Transfection Reagent TransIT -LT1 Transfection Reagent CELL LINE SPECIFIC TransIT -293 Transfection Reagent... 1 TransIT -3T3 Transfection Kit... 1 NEW! TransIT -BrCa Transfection Reagent...1 & 12 TransIT -CHO Transfection Kit... 1 TransIT -COS Transfection Kit... 1 TransIT-HeLaMONSTER Transfection Kit TransIT -Jurkat Transfection Reagent TransIT -Keratinocyte Transfection Reagent TransIT-Neural Transfection Reagent TransIT -Prostate Transfection Kit LARGE SCALE PROTEIN PRODUCTION TransIT-PRO Transfection Kit DIMENSIONAL CELL GROWTH TransIT -3D Transfection Reagent/3D Transfection System HIGH THROUGHPUT TransIT -Express Transfection Reagent OLIGONUCLEOTIDE TransIT -Oligo Transfection Reagent PLASMID CONTROLS Plasmid Delivery Control, Cy Plasmid Delivery Control, Fluorescein RNA TRANSFECTION sirna/mirna TransIT-TKO Transfection Reagent TransIT-siQUEST Transfection Reagent LARGE RNA (Viral RNA and mrna) TransIT -mrna Transfection Kit RNAi Controls RNAi Delivery Control, Cy RNAi Delivery Control, Fluorescein ELECTROPORATION PLASMID DNA, RNA, sirna and mirna Ingenio Electroporation Kit Ingenio Electroporation Solution Ingenio Electroporation Accessories TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com

4 DNA/siRNA Transfection Broad Spectrum DNA & sirna/mirna NEW! TransIT-X2 DYNAMIC DELIVERY SYSTEM Efficient Exceptional broad spectrum transfection Versatile Cutting edge delivery of plasmid DNA and sirna/mirna Technology Novel, non-liposomal, polymeric delivery PRODUCT NO. MIR 3 MIR 4 MIR MIR 5 MIR 6 QUANTITY.3 ml.75 ml 1.5 ml 5 x 1.5 ml 1 x 1.5 ml To inquire about bulk pricing, please call (toll free in the U.S.) or We recently tested the TransIT-X2 Dynamic Delivery System headto-head against Lipofectamine for DNA transfection of NIH-3T3 fibroblasts and the breast cancer cell line ZR We observed higher efficiency and less toxicity when using TransIT-X2. We are also pleased to hear that TransIT-X2 will be offered in similar volume configurations to Lipofectamine. Dr. Edwin Li, Assistant Professor Saint Joseph s University Description Achieve superior transfections with an innovative polymeric system that efficiently delivers both DNA and RNA out of the endosome and into the cytoplasm, overcoming a critical barrier to nucleic acid delivery. TransIT-X2 TM Dynamic Delivery System A549 CHO-K1 Hep G2 HCC 1143 HUVEC LNCaP MDA-MB-468 MDCK HMEC (epithelial) T47D Immortalized Keratinocytes FreeStyle TM 293-F BT- NHDF NIH-3T3 HEK 293 PC-3 PC-12 HCC38 SK-N-MC AU565 COS-7 HeLa MCF-7 MDA-MB-231 RAW Lipofectamine Keratinocytes Caco-2 MDA-MB-453 SH-SY5Y FIGURE 1. TransIT-X2 Dynamic Delivery System Enables Superior Gene Expression in a Variety of Cell Types. TransIT-X2 Dynamic Delivery System and Lipofectamine Transfection Reagent were used to transfect plasmid DNA encoding luciferase into 3 different cell types at three reagent-to-dna ratios. Luciferase expression was compared at 24 hours post-transfection using a standard luciferase assay. Head-to-head comparisons at optimized ratios illustrate superior or equal luciferase expression using TransIT-X2 in 26 of 3 cell types; 11 cell types had expression levels 2-fold higher than Lipofectamine (denoted with ). 2 Cell types with >2-fold luciferase expression in head-to-head comparisons. DNA/siRNA Transfection >>

5 TransIT-X2 Dynamic Delivery System continued A549 CHO-K1 LNCaP MDCK Hep G2 DNA/siRNA Transfection PC-12 HMEC* NHDF* - FIGURE 2. Visualization of High GFP Expression Using TransIT-X2 Dynamic Delivery System. TransIT-X2 Dynamic Delivery System was used to transfect plasmid DNA encoding EGFP into A549, CHO-K1, Hep G2, LNCaP, MDCK, PC-12, primary human mammary epithelial cells (HMEC) and primary normal human dermal fibroblasts (NHDF). Transfections were performed in 35 mm MatTek dishes using 4-8 μl of TransIT-X2 to deliver 2 μg of DNA. Images (32X) were captured at 48 hours post-transfection using a Zeiss Axiovert S inverted fluorescence microscope. *Indicates primary cell types. EGFP Positive (%) A549 CHO-K1 Hep G2 MDCK LNCaP FIGURE 3. High GFP Transfection Efficiency in Multiple Cell Lines and Primary Cells Using TransIT-X2 Dynamic Delivery System. TransIT-X2 Dynamic Delivery System was used to transfect plasmid DNA encoding EGFP into A549, CHO-K1, Hep G2, MDCK, LNCaP, PC-12, primary human mammary epithelial cells (HMEC) and primary normal human dermal fibroblasts (NHDF). Transfections were performed in 96-well plates using.2-.4 μl of TransIT-X2 to deliver.1 μg of DNA (2:1, 3:1 or 4:1 reagent: DNA ratio). Triplicate wells were assayed 48 hours post-transfection on a guava easycyte 5HT Flow Cytometer. *Indicates primary cell types. TheTransfectionExperts.com PC-12 HMEC* NHDF* For Technical Support Tel techsupport@mirusbio.com 3

6 DNA/siRNA Transfection TransIT-X2 Dynamic Delivery System continued FIGURE 4. Functional Co-delivery of Plasmid DNA and sirna Using TransIT-X2 Dynamic Delivery System. TransIT-X2 Dynamic Delivery System was used to transfect plasmid Cy 5 labeled DNA encoding nuclear YFP and Cy 3 labeled sirna into HeLa cells. Transfection was performed in a 6-well plate with Poly-L-Lysine (PLL) coated coverslips using 4 μl of TransIT-X2 to deliver 2 μg of DNA (2:1 reagent:dna ratio) and 25 nm sirna. Actin cytoskeleton was stained using Alexa Fluor 35 Phalloidin. Image (63X) was captured at 24 hours post-transfection using a Nikon A1R confocal microscope. Image key: yellow (nuclear YFP), blue (Cy5 labeled DNA), red (Cy3 labeled sirna), green (actin cytoskeleton). We work on non-small cell lung cancer (NSCLC) which is an adherent cell culture line. Previously, we have tested many transfection products from several companies without much success, but the TransIT-X2 Dynamic Delivery System works very well with NSCLC using my protocol. Dr. Luo Wang, University of Michigan Comprehensive Cancer Center The TransIT-X2 Dynamic Delivery System outperformed all other transfection reagents we have tested for DNA transfection of our C2C12 mouse myoblast cell line. In addition, TransIT-X2 was also less toxic. Dr. G. Du, Assistant Professor Texas Medical Center We are pleased with the performance of the TransIT-X2 Dynamic Delivery System when transfecting our renal carcinoma cell line Sathish Padi, North Dakota State University The TransIT-X2 Dynamic Delivery System performed better than our regular transfection reagent (Polyjet) for delivering DNA into the hard to transfect A549 cell line. TransIT-X2 was able to show protein expression compared to Polyjet which failed to produce detectable levels of protein containing V5 tag. Jason Liggett and Kyung-Won Min, Baek Lab University of Tennessee 4 DNA/siRNA Transfection >>

7 TransIT-X2 Dynamic Delivery System continued Knockdown (%) Transfection Reagent sirna Target TransIT-X2 Lipofectamine GAPDH TransIT-X2 Lipofectamine aha1 DNA/siRNA Transfection FIGURE 5. TransIT-X2 Dynamic Delivery System Achieves Higher Knockdown than Lipofectamine. Human TransIT-X2 Dynamic Delivery System and Lipofectamine Transfection Reagent were used to transfect sirna targeting endogenous proteins - GAPDH and aha1 or to deliver a non-targeting control in primary normal human dermal fibroblasts (NHDF). Cells were transfected in a 6-well plate using 4 μl of TransIT-X2 or 6 μl of Lipofectamine and 25 nm sirna according to each manufacturer's protocol. The amount of GAPDH or aha1 mrna was measured relative to 18s rrna levels using qrt-pcr and then scaled to the mrna levels of the negative control, 48 hours post-transfection. Error bars represent the standard deviation of triplicate wells. Knockdown of PTK9 mrna (%) Transfection Reagent mirna TransIT-X2 Lipofectamine Pre-miR hsa-mir-1 TransIT-X2 Lipofectamine mirvana mimic, mir-1 FIGURE 6. Effective mirna Delivery Using TransIT-X2 Dynamic Delivery System Yields Decreased Levels of PTK9 mrna. TransIT-X2 Dynamic Delivery System and Lipofectamine Transfection Reagent were used to transfect T47D cells with Pre-miR hsa-mir-1 mirna Precursor or mirvana mirna mimic, mir-1, both known to decrease PTK9 mrna levels. A Pre-miR negative control was transfected to assess baseline mrna levels. Cells were transfected in a 12-well plate using 3 μl of TransIT-X2 or Lipofectamine and 5 nm mirna according to each manufacturer's protocol. The amount of PTK9 mrna was measured relative to 18s rrna levels using qrt-pcr and then scaled to the mrna levels of the negative control, 48 hours post-transfection. Error bars represent the standard deviation of triplicate wells. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 5

8 DNA Transfection Broad Spectrum DNA TransIT - TRANSFECTION REAGENT Broad Spectrum DNA Delivery Achieve high expression in many cell types, including hardto-transfect, insect, stem and primary cells Outperforms Competitor Reagents TransIT- demonstrates higher protein yield and less toxicity when compared to other transfection reagents Animal Origin Free provides high performance with maximum compatibility PRODUCT NO. MIR 54 MIR 5 MIR 55 MIR 56 QUANTITY.4 ml 1. ml 5 x 1. ml 1 x 1. ml To inquire about bulk pricing, please call (toll free in the U.S.) or Description TransIT- Reagent is a versatile transfection solution for broad spectrum DNA delivery into mammalian cells. This premium reagent is animal component free allowing maximum compatibility for all downstream applications while outperforming major competitors in most cell types. A. B. IDEAL FOR USE IN VIRUS PRODUCTION FIGURE 7. TransIT - Reagent Efficiently Transfects Human Induced Pluripotent Stem (ips) Cells. TransIT- Transfection Reagent was used to transfect.5 x 1 6 ips cells with a ZsGreen expressing plasmid (Clontech). Transfections were performed in 6-well plates using 7.5 µl of TransIT- Transfection Reagent to deliver 2.5 µg of DNA (3:1, reagent: DNA). Cells were visualized 24 hours post-transfection and imaged at 4X objective with an Olympus IX71 Inverted Microscope. Image is (A) green fluorescence. Cells were also assayed 24 hours post-transfection on an Accuri Cytometer. The histogram (B) shows untransfected cells (black line) compared to cells transfected with plasmid using TransIT- (green line). Data courtesy of Cellular Dynamics International. Luciferase (RLU) 6 9, 7, 7,, 5,, 3,,, 2:1 3:1 4:1 1.5:1 3:1 5:1 1.5:1:1 3:1:1 5:1:1 TransIT - Lipofectamine Lipofectamine LTX and PLUS TM Toxicity (%) FIGURE 8. TransIT - Reagent Exhibits Higher Expression and Lower Cellular Toxicity Compared to Other Transfection Reagents. Human umbilical vein endothelial cells (HUVEC) were transfected with a luciferase expression plasmid using the designated reagents at the reagent-to- DNA ratios indicated beneath each bar. Transfections were performed in 96-well plates using.1 µg of plasmid DNA per well. Luciferase expression (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as percent cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph. Error bars represent the standard deviation of triplicate wells. DNA Transfection >>

9 TransIT - Transfection Reagent continued Normalized Luciferase Expression (%) HUVEC THP-1 NT2 CHO-K1 HEK-293 TransIT - FuGENE HD Lipofectamine FIGURE 9. Superior Gene Expression in a Broad Spectrum of Cell Types. The indicated cell types were transfected in 96-well plates with a luciferase expression plasmid (.1 μg/well) according to industry accepted testing protocols. Reagent-to-DNA ratios were optimized for each cell type: TransIT - (Mirus Bio, 2:1 or 3:1), FuGENE HD (Promega, 3.5:1), Lipofectamine (Life Technologies, 1.5:1, 3:1 or 5:1). Luciferase activity was measured 24 hours posttransfection. Values were normalized to TransIT- and presented as a percentage of luciferase expression. FuGENE is a registered trademark of Fugent LLC. Lipofectamine is a registered trademark of Life Technologies Corporation. DNA Transfection 2.5 Relative Levels of Secreted Alkaline Phosphatase (as determined by O.D. 5 nm) Effectene (optimized 5:1;.4 ug) FectoFly (1:1;.4 ug) FectoFly (1:1;.8 ug) FectoFly (1:1; 1.2 ug) FectoFly (1:1; 3. ug) FlyFectin (2:1;.4 ug) FlyFectin (4:1;.4 ug) FlyFectin (8:1;.4 ug) FlyFectin (16:1;.4 ug) FuGENE HD (3:2;.4 ug) FuGENE HD (5:2;.4 ug) FuGENE HD (7:2;.4 ug) FuGENE HD (8:2;.4 ug) Insect GeneJuice (5:1;.4 ug) Transfection Condition FIGURE 1. TransIT - Reagent Effectively Transfects Drosophila S2 Cells. Cells were transfected with a plasmid construct expressing a secreted form of the Dscam extracellular domain fused to alkaline phosphatase (AP) in a 24- well plate. The ratio of transfection reagent-to-dna and micrograms of DNA per well is noted beneath each bar. All products were used according to manufacturers protocol. All transfections were performed in serum-free media for 4 hours followed by complete media supplementation. An AP enzymatic assay was used to measure the AP levels 24 hours post-transfection. TheTransfectionExperts.com Insect GeneJuice (1:1;.4 ug) Insect GeneJuice (15:1;.4 ug) Insect GeneJuice (:1;.4 ug) Linear PEI 25 kd (5:1;.4 ug) Linear PEI 25 kd (1:1;.4 ug) Linear PEI 25 kd (15:1;.4 ug) Linear PEI 25 kd (:1;.4 ug) PromoFectin-Insect (5:1;.4 ug) PromoFectin-Insect (1:1;.4 ug) PromoFectin-Insect (15:1;.4 ug) PromoFectin-Insect (:1;.4 ug) TransIT- (4:1;.4 ug) TransIT- (6:1;.4 ug) TransIT- (8:1;.4 ug) TransIT- (1:1;.4 ug) Data courtesy of a Researcher, University of California, Berkeley. For Technical Support Tel techsupport@mirusbio.com 7

10 Broad Spectrum DNA TransIT -LT1 TRANSFECTION REAGENT DNA Transfection Broad Spectrum DNA Delivery Utilize one transfection reagent and protocol for a variety of cells Low Cellular Toxicity Maintain cell density and reduce experimental biases Deliver Single or Multiple Plasmids Suitable for many applications such as gene expression, shrna expression, virus production and promoter analysis PRODUCT NO. MIR 234 MIR 23 MIR 235 MIR 236 QUANTITY.4 ml 1. ml 5 x 1. ml 1 x 1. ml To inquire about bulk pricing, please call (toll free in the U.S.) or We routinely use Mirus TransIT -LT1 Transfection Reagent for the delivery of plasmid DNA to carry out immunoprecipitation experiments. Our lab recently published using TransIT -LT1 for this application to reveal a crucial regulator (MCUR1) for calcium uptake in the mitochondria to regulate cellular metabolism. (Mallilankaraman, K et al. Nature Cell Biology. December 12). Dr. Karthik Mallilankaraman, Madesh Laboratory, Center for Translational Medicine, Temple University Description TransIT -LT1 (Low Toxicity) Reagent is a broad spectrum, high efficiency DNA transfection reagent that is easy to use and exhibits minimal cellular toxicity. This reagent is a proprietary formulation of polyamines and cationic lipids that efficiently transfects cells in the presence of serum. 2,5, IDEAL FOR USE IN VIRUS PRODUCTION Luciferase (RLU) 2,, 1,5, 1,, 5, Cytotoxicity (%) 8 Cells Alone 2:1 3:1 4:1 1.5:1 3:1 5:1 1.5:1 3:1:1 5:1:1 TransIT -LT1 Lipofectamine Lipofectamine LTX and PLUS 1.5:1 2:5:1 3:5:1 FuGENE HD FIGURE 11. TransIT -LT1 Reagent Exhibits Higher Expression and Lower Cellular Toxicity Compared to Other Transfection Reagents. Hep G2 cells were transfected with a luciferase expression plasmid using the designated reagents at the manufacturers' recommended reagent-to-dna ratio indicated beneath each bar. Transfections were performed in 96-well plates using.1 µg of plasmid DNA per well. Luciferase expression (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as percent cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph. Experiments were performed as per industry accepted testing protocols. FuGENE is a registered trademark of Fugent LLC. Lipofectamine is a registered trademark of Life Technologies Corporation. DNA Transfection >>

11 TransIT -LT1 Transfection Reagent continued EGFP Expressing Cells (%) A549 CHO-K1 COS-7 HEK 293 HeLa Hep G2 Keratinocytes LNCaP Neuro-2a NIH 3T3 PC-3 FIGURE 12. TransIT -LT1 Reagent Efficiently Delivers DNA to a Wide Variety of Cell Lines. Using TransIT-LT1 Transfection Reagent, cells were transfected with the pegfp-c1 expression vector, and the percentage of EGFP expressing cells was determined hours post-transfection by flow cytometry. 1.E + 8 DNA Transfection Luciferase Activity (RLU) 1.E E E + 5 CHO-K1 COS-7 HEK 293 HeLa TransIT -LT1 FuGENE 6 FIGURE 13. Comparable Luciferase Expression with TransIT -LT1 Reagent and FuGENE 6 in Multiple Cell Types. The indicated cell lines were transfected in duplicate with 1 µg of a luciferase expression vector per well of a 12-well plate using either 3 µl of the TransIT-LT1 or FuGENE 6 Reagents according to industry accepted testing protocols. Cells were harvested 24 hours post-transfection and assayed for luciferase activity. FuGENE is a registered trademark of Fugent LLC. TheTransfectionExperts.com FIGURE 14. High Efficiency Transfection of icell Cardiomyocytes using TransIT -LT1 Transfection Reagent. icell Cardiomyocytes were plated at, cells/well in a 96-well tissue culture plate coated with.1% gelatin. After allowing the cells to recover from thaw, cells were transfected with ng/well of pmaxgfp (Lonza) using TransIT-LT1 Transfection Reagent with a 2:1 reagent-to- DNA ratio according to the manufacturer s instructions. Fluorescent images were taken 3 days post-transfection using a Olympus IX71 inverted microscope. Data courtesy of Cellular Dynamics International. For Technical Support Tel techsupport@mirusbio.com 9

12 DNA Transfection Cell Line Specific TransIT CELL LINE SPECIFIC TRANSFECTION REAGENTS TransIT Cell Line Specific DNA Transfection Reagents are formulated to maximize transfection efficiency while maintaining cellular health in many popular or hard-to-transfect cell types. All of these reagents offer: Optimized Formulations Designed for each cell type Low Cellular Toxicity Maintain cell density and reduce experimental biases due to toxicity-induced cellular changes Serum Compatible No media changes necessary or extensive optimization required, saving valuable research time Product* Applicable Cell Line(s) or Cell Type(s) TransIT -293 Transfection Reagent HEK 293, HEK 293T, 75 85% and related To inquire about bulk pricing, please call (toll free in the U.S.) or Efficiency** Product No. Quantity MIR 274 MIR 27 MIR 275 MIR 276 TransIT -3T3 Transfection Kit (TransIT -3T3 & 3T3 Authority Reagents) NIH 3T3, 3T3-L1 and related TransIT -BrCa Transfection Reagent IDEAL FOR USE IN VIRUS PRODUCTION 55 65% MIR 2184 MIR 21 MIR 2185 MIR ml 1. ml 5 x 1. ml 1 x 1. ml.4 ml 1. ml 5 x 1. ml 1 x 1. ml MCF-7, MDA-MB-231, MDA- MB-453, MDA-MB-468, T47D % MIR 554 MIR 55 MIR 555 MIR ml 1. ml 5 x 1. ml 1 x 1. ml TransIT -CHO Transfection Kit (TransIT -CHO Reagent & CHO Mojo Reagent) MIR ml MIR ml CHO-K1 and related 5 % MIR x 1. ml MIR x 1. ml TransIT -COS Transfection Kit (TransIT -COS Reagent & COS Boss Reagent) MIR ml MIR ml COS-1, COS-7 and related >% MIR x 1. ml MIR x 1. ml 1 DNA Transfection >>

13 Product* Applicable Cell Line(s) or Cell Type(s) Efficiency** Product No. Quantity TransIT-HeLaMONSTER Transfection Kit (TransIT -HeLa Reagent and MONSTER Reagent) TransIT -Jurkat Transfection Reagent HeLa and related 5 % Jurkat, Jurkat-E6, RAW 264.7, THP-1, K562, and other lymphoid cell lines TransIT -Keratinocyte Transfection Reagent TransIT-Neural Transfection Reagent 1 25% Immortalized Keratinocyte 3% C6, Daoy, DB-TRG-5MG, DI-TNC1, HCN-1A, Neuro-2a, PC-12, SK-N-MC, SVGp12 >75% MIR 294 MIR 29 MIR 295 MIR 296 MIR 2124 MIR 21 MIR 2125 MIR 2126 MIR 24 MIR 2 MIR 25 MIR 26 MIR 2144 MIR 21 MIR 2145 MIR ml 1. ml 5 x 1. ml TransIT -Prostate Transfection Kit (TransIT -Prostate Reagent and Prostate Boost Reagent) DU 145, LNCAP, PC-3 >5% MIR 2134 MIR 213 MIR 2135 MIR x 1. ml.4 ml 1. ml 5 x 1. ml 1 x 1. ml.4 ml 1. ml 5 x 1. ml 1 x 1. ml.4 ml 1. ml 5 x 1. ml 1 x 1. ml.4 ml 1. ml 5 x 1. ml 1 x 1. ml DNA Transfection * Single tube reagents contain the indicated transfection reagent. Transfection reagents with two components are named Kits and both components are listed following the product name. ** Transfection efficiency determined by transfection of an EGFP expression vector followed by visual quantification of the percentage of cells expressing EGFP or via flow cytometry. Our lab has been satisfied with the routine use of the TransIT-HelaMONSTER Transfection Kit. Transfections exhibit high target protein expression with very little cell toxicity. Cells remain viable post-transfection and can be readily infected with virus without any problems. Dr. Corine St. Gelais, The Ohio State University Center for Retrovirus Research TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 11

14 DNA Transfection Cell Line Specific NEW! TransIT -BrCa TRANSFECTION REAGENT Superior DNA Delivery Achieve high expression levels in breast cancer cell types including: MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468 and T47D Formulated for Low Cellular Toxicity Maintain cell density and reduce experimental biases due to toxicity PRODUCT NO. MIR 554 MIR 55 MIR 555 MIR 556 QUANTITY.4 ml 1. ml 5 x 1. ml 1 x 1. ml To inquire about bulk pricing, please call (toll free in the U.S.) or Description TransIT -BrCa Transfection Reagent is the only breast cancer cell line reagent available to the market today. While it delivers efficiently to many breast cancer cell types, it also maintains low cellular toxicity resulting in reduced experimental biases. EGFP Positive (%) MCF-7 MDA-MB-231 MDA-MB-453 MDA-MB-468 T47D FIGURE 15. TransIT -BrCa Transfection Reagent Yields High Efficiency Plasmid DNA Transfection. TransIT-BrCa Transfection Reagent was used to transfect plasmid DNA encoding GFP into MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468 and T47D breast cancer cell lines. Transfections were performed in 24-well plates using µl of TransIT-BrCa Transfection Reagent to deliver.5 µg of DNA (2:1 and 3:1, reagent:dna ratio). Triplicate wells were assayed 48 hours post-transfection on a guava easycyte 5HT flow cytometer. 12 DNA Transfection >>

15 Luciferase (RLU),, 3,,,, 1,, TransIT -BrCa Transfection Reagent continued MCF-7 Luciferase (RLU),, 15,, 1,, 5,, MDA-MB-231 2:1 3:1 4:1 2:1 3:1 4:1 2:1 3:1 4:1 TransIT -BrCa Lipofectamine FuGENE HD 2:1 3:1 4:1 2:1 3:1 4:1 2:1 3:1 4:1 TransIT -BrCa Lipofectamine FuGENE HD,, 15,, Luciferase (RLU) 5,, MDA-MB-453 4,, 3,, 2,, 1,, 2:1 3:1 4:1 2:1 3:1 4:1 2:1 3:1 4:1 TransIT -BrCa Lipofectamine FuGENE HD MDA-MB-468,, 15,, T47D DNA Transfection Luciferase (RLU) 1,, 5,, Luciferase (RLU) 1,, 5,, 2:1 3:1 4:1 2:1 3:1 4:1 2:1 3:1 4:1 TransIT -BrCa Lipofectamine FuGENE HD 2:1 3:1 4:1 2:1 3:1 4:1 2:1 3:1 4:1 TransIT -BrCa Lipofectamine FuGENE HD FIGURE 16. TransIT -BrCa Transfection Reagent Exhibits Higher Luciferase Expression in Breast Cancer Cells Compared to Other Transfection Reagents. Breast Cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468 and T47D, were transfected with a luciferase expression plasmid using the designated reagents at the reagent-to-dna ratios indicated beneath each bar according to industry accepted protocols. Transfections were performed in 96-well plates using.1 µg of plasmid DNA per well. Luciferase expression was measured at 24 hours post-transfection using a standard assay. Error bars represent the standard deviation of triplicate wells. Relative Luciferase Units 3 1 E2 1nM 1nM 1nM 1nM Negative Control ERE Reporter Plasmid MCF-7 (ER positive) MDA-MB-231 (ER negative) FIGURE 17. Activation of Estrogen Receptor Pathway is Detected in MCF-7 Cells Transfected with the TransIT -BrCa Transfection Reagent. MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) breast cancer cells were maintained in complete media containing charcoal stripped FBS for 3 days prior to transfection. TransIT-BrCa Transfection Reagent was used to deliver an ERE-luciferase reporter plasmid or negative control (SA Biosciences) at a 2:1 reagent-to-dna ratio. Transfections were performed in 96- well plates using.1 µg of plasmid DNA per well. Twenty-four hours post-transfection cells were treated with varying levels of 17-estradiol (E2) for 6 hours. Firefly and Renilla luciferase expression was measured at 3 hours post-transfection using a standard assay. Promoter activity is represented as the ratio of firefly to luciferase relative light units (RLU) using the Renilla luciferase for normalization. Error bars represent the standard deviation of triplicate wells. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 13

16 DNA Transfection Large Scale Protein Production TransIT-PRO TRANSFECTION KIT High Performance Achieve high protein yield in suspension CHO and 293 cell types Easy to Use Compatible with multiple CHO media formulations Total Cost Savings Higher protein yield translates to lower material and labor costs compared to linear PEI PRODUCT NO. MIR 57 MIR 57 QUANTITY 1 ml 1 ml To inquire about bulk pricing, please call (toll free in the U.S.) or We recently engineered a bispecific immunofusion for the treatment and elimination of leukemia stem cells. For this work we chose TransIT-PRO for antibody production of CHO-S cells based on the high protein yield we obtained. (Kuo et al, Protein Eng Des Sel. Oct 12). Jen-Sing Liu, Ph.D., Molecular Templates Inc. Description Decrease time to produce usable protein by maximizing target protein yields through transient transfection. The TransIT-PRO Transfection Kit uses animal origin free components designed for high and reproducible protein yield in suspension CHO and 293 derived cells. Since it is compatible with varied media formulations, the same media can be used for both transient and stable expression. The TransIT-PRO outperforms linear PEI in protein yield, while providing a cost-effective alternative to FreeStyle MAX. A. Human Fc (µg/l) 3 Day 3 Day 5 Day 7 TransIT-PRO + 25kDa linear PEI PRO Boost Reagent FreeStyle MAX B. PRO PEI FS S1 S2 S3 higg1 FIGURE 18. Achieve High Antibody Titers Using TransIT-PRO Transfection Kit in Suspension CHO Cells. IgG1 was produced by transient transfection using TransIT-PRO and PRO Boost Reagent (1:1:1), 25 kda linear PEI (6:1) or FreeStyle MAX (1:1) transfection reagents according to the manufacturers' or published protocol (reagent:dna ratio). Transfections were performed using 1 µg plasmid DNA per milliliter of culture and.5 x 1 6 cells/ml at the time of transfection. FreeStyle CHO-S cells were cultured in ml of FreeStyle CHO Expression medium in 125 ml shake flasks. (A) Day 3, 5 and 7 supernatants were clarified and analyzed using a human IgG-Fc sandwich ELISA. Error bars represent the standard deviation of triplicate technical replicates, 25kDa linear PEI is duplicate technical replicates. (B) Day 7 supernatants were clarified and analyzed by Western blot. An IgG standard was included for quantification estimate (S1= 1.6 mg/l, S2= 3.2 mg/l, S3 = 6.3 mg/l). 14 DNA Transfection >>

17 TransIT-PRO Transfection Kit continued 175 Human Fc (µg/l) BD Select TM CD Medium BD Select TM CHO Medium FreeStyle TM CHO Expression Medium PowerCHO 2 CD Medium ProCHO TM 5 Medium 5 25 FIGURE 19. TransIT-PRO Provides High Performance Across Varied Media Formulations. FreeStyle CHO-S cells were adapted to five representative growth media as noted in the graph. Cells were transfected with an IgG encoding plasmid using the TransIT-PRO and PRO Boost Reagent (1:1:1), 25 kda linear PEI (6:1) (Polysciences), or FreeStyle MAX (1:1) (Life Technologies Corporation) transfection reagents according to published protocol (reagent:dna ratio). Transfections were performed in 24-well deep well shaker blocks using 1 µg plasmid DNA per milliliter of culture and.5 x 1 6 cells/ ml at the time of transfection. Human IgG1 was quantitated from day 5 clarified supernatants and analyzed by a human anti-fc sandwich ELISA. Error bars represent the standard deviation of triplicate wells. Yield (mg/l) TransIT-PRO + PRO Boost Reagent 25kDa Linear PEI FreeStyle TM Max DNA Transfection Protein TransIT-PRO Transfection Reagent (Mirus Bio) 293Fectin (Life Technologies) FIGURE. Achieve High Protein Yields Using TransIT-PRO Transfection Kit in Suspension 293 Cells. Ten different secreted (non antibody) proteins were transiently expressed in FreeStyle 293 F cells (Life Technologies) using the TransIT PRO (1.5:1) or 293fectin (Life Technologies, 2:1) transfection reagents according to manufacturers protocol. Cells were grown in FreeStyle 293 Expression Medium and transfected at a density of 4 x 1 6 cells/ml. The scale of the transfection for each protein varied between 1 6 L of culture. Data courtesy of a TransIT PRO pharmaceutical customer. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 15

18 DNA Transfection 3-Dimensional Cell Growth TransIT -3D TRANSFECTION REAGENT High Efficiency DNA Delivery Achieve high expression in a wide range of cell types grown in 3D culture Easy-to-use User friendly protocol that requires minimal optimization Flexibility Compatible with solid matrix scaffold and hydrogel 3D culture formats PRODUCT NO. QUANTITY MIR 54.4 ml MIR 5 1. ml MIR 55 5 x 1. ml MIR 56 1 x 1. ml 3D Transfection System Includes:.4 ml TransIT-3D Transfection Reagent and 2 alvetex 3D 12-well Plates MIR 58 Description Ideal for the transfection of cells in 3D cell culture, the 3D Transfection System combines the technologies of Reinnervate's alvetex 3D Cell Culture Plates and Mirus Bio's TransIT -3D Transfection Reagent, enabling scientists to create models that more accurately mimic the tissue environment, gaining a much deeper insight into the complexities of cell function and behavior. each To inquire about bulk pricing, please call (toll free in the U.S.) or ,5,, 1,25,, 1,,, Luciferase Activity (RLUs) 75,, 3,, 25,,,, 15,,,, 5,, Reagent-to-DNA Ratio 2:1 3:1 2:1 3:1 2:1 CHO-K1 HeLa HepG2 MCF-7 NIH-3T3 FIGURE 21. 3D Transfection of Multiple Cell Types. The indicated cell lines were seeded at optimized cell densities in 12-well alvetex 3D plates and adapted to 3D culture conditions for 48 hours. Post-adaptation, cells were transfected with TransIT -3D combined with a plasmid encoding firefly luciferase at the reagent-to-dna ratios indicated. Luciferase activity was measured 24 hours post-transfection using a conventional assay. FIGURE 22. GFP Expression in 3D Culture. NIH 3T3 fibroblast cells were seeded in an alvetex 12-well plate and adapted to 3D growth. Forty-eight hours post-adaptation, cells were transfected with TransIT -3D combined with a plasmid encoding Green Fluorescent Protein (GFP). Cells were counterstained with the nuclear stain Hoechst (blue) and visualized via confocal microscopy. Data courtesy of Reinnervate. 16 DNA Transfection >>

19 High Throughput TransIT -EXPRESS TRANSFECTION REAGENT High Throughput Reagent Designed and optimized specifically for reverse transfections Broad Spectrum DNA Delivery Utilize one transfection reagent and protocol for a variety of cells Low Cellular Toxicity Maintain cell density and reduce experimental biases PRODUCT NO. MIR 4 MIR MIR 5 MIR 6 QUANTITY.4 ml 1. ml 5 x 1. ml 1 x 1. ml To inquire about bulk pricing, please call (toll free in the U.S.) or Description TransIT-Express Transfection Reagent is a broad spectrum, low toxicity DNA transfection reagent optimized for high-throughput 96-well plate transfections. This reagent is compatible with standard transfections of adherent cells or increasingly popular reverse transfections. FIGURE 23. TransIT -Express Reagent is Designed and Optimized Specifically for High-throughput and Reverse Transfections. DNA Transfection Oligonucleotide TransIT -OLIGO TRANSFECTION REAGENT Unique Formulation Maximize DNA and RNA oligonucleotide transfection efficiency in a wide range of cells PRODUCT NO. MIR 2164 MIR 21 MIR 2165 MIR 2166 QUANTITY.4 ml 1. ml 5 x 1. ml 1 x 1. ml To inquire about bulk pricing, please call (toll free in the U.S.) or Oligonucleotides Tested phosphodiester DNA, phosphothioate DNA (sdna), phosphothioate RNA (srna), 2'OMe RNA, 2'OMe RNA/sDNA Chimerics, and Morpholino/DNA duplexes. FIGURE 24. TransIT -Oligo Reagent Achieves High Transfection Efficiency. HeLa cells were transfected with Cy 3 and fluorescein labeled phosphothioate DNA oligos using TransIT-Oligo Reagent and observed 24 hours post-transfection. The yellow punctate fluorescence is the result of co-localization of the fluorescein and Cy 3 labeled oligonucleotides after transfection. The image was acquired using a confocal microscope. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 17

20 DNA Transfection Plasmid Controls Label IT PLASMID DELIVERY CONTROLS Chemically Labeled Using Mirus established covalent Label IT technology Sensitive Directly track plasmid DNA delivery using fluorescent microscopy Inert and Compatible Noncoding controls that are suitable for co-delivery experiments with other functional plasmids Ready-to-Use Supplied as.5 mg/ml prelabeled plasmid DNA control solution for in vitro and in vivo tracking studies LABEL PRODUCT NO. QUANTITY Cy 3 MIR µg MIR 795 µg Fluorescein MIR µg MIR 797 µg Description Label IT Plasmid Delivery Controls consist of either Cy 3 or fluorescein labeled 2.7 kb noncoding plasmid for direct assessment of delivery efficiency in mammalian cells. Covalent Bond Labeled Plasmids FIGURE 25. Ready-to-use Label IT Plasmid Delivery Controls are Labeled Using the Covalent Label IT Technology. Mirus Label IT reagents* are used to chemically attach labels to a noncoding plasmid at optimized label/base pair ratio to generate Label IT Plasmid Delivery Controls. These pre-labeled Label IT Plasmid Delivery Controls facilitate direct tracking of plasmid DNA delivery by fluorescence microscopy for in vitro and in vivo studies. The image shows HeLa cells transfected in serum-containing media with the Label IT Cy 3 Plasmid Delivery Control (red) using the TransIT -LT1 Transfection Reagent. Twenty-four hours post-transfection, the cells were fixed, then counterstained to locate the nuclei (blue) and to stain the actin (green). *Label IT kits for a wide variety of applications are also available for purchase. Visit mirusbio.com/labeling for more information. 18 Transfect Labeled Plasmid and Detect by Fluorescence Microscopy RNA DNA Transfection >>

21 sirna/mirna TransIT-TKO & TransIT-siQUEST TRANSFECTION REAGENTS High Knockdown Efficiency Achieve optimal gene silencing in a large percentage of cells to ensure experimental success Low Cellular Toxicity Maintain cell density and reduce experimental biases due to alterations in cellular health Flexible Protocol use with either standard or reverse transfections TransIT-TKO Transfection Reagent PRODUCT NO. QUANTITY MIR ml MIR ml MIR x 1.5 ml MIR x 1.5 ml TransIT-siQUEST Transfection Reagent PRODUCT NO. QUANTITY MIR ml We have tried other transfection reagents, but MIR ml only the TransIT -TKO reagent gives us a % transfection rate and gene knockdown without MIR x 1.5 ml toxicity in these cells (RAW 264.7). Nature Protocols, MIR x 1.5 ml 1: (6) To inquire about bulk pricing, please call (toll free in the U.S.) or Description TransIT-TKO and TransIT-siQUEST small interfering RNA (sirna and mirna) Transfection Reagents are broad spectrum reagents that are easy to use and exhibit minimal cellular toxicity. Each reagent is uniquely formulated and exhibits distinct sirna/mirna transfection profiles. These two reagents allow the user to identify the best transfection reagent for their particular cell line. Luciferase Expression (%) 3 1 TransIT-siQUEST Reagent TransIT-TKO Reagent Lipofectamine RNA Transfection Reagent Alone CHO-K1 COS-7 HEK 293 HepG2 TheTransfectionExperts.com MCF-7 NIH 3T3 RAW264.7 FIGURE 26. Knockdown Efficiencies Using TransIT-siQUEST, TransIT-TKO Reagents and Lipofectamine. Firefly and sea pansy luciferase reporter vectors were co-transfected into various cell lines using TransIT -LT1 Reagent. Subsequently, firefly luciferase expression was knocked down by transfection of 25 nm anti-firefly luciferase sirna using either TransIT-siQUEST (red), TransIT-TKO (tan) or Lipofectamine (gray) Reagents. Bars indicate the percent of normalized firefly luciferase expression as compared to each reagent alone control 24 hours posttransfection. For Technical Support Tel techsupport@mirusbio.com Vero 19

22 TransIT-TKO & TransIT-siQUEST Transfection Reagents continued DNA Transfection Cell Line (Source) Endogenous Transcript TransIT-TKO Knockdown Efficiency TransIT-siQUEST Knockdown Efficiency A549-luc (human lung) Luciferase* 77% 82% BNL CL.2 MAPK1 % -- (mouse liver) MAPK3 83% -- CHO-luc (hamster ovary) Luciferase* 86% 91% HEK 293-lux (human kidney) Luciferase* 83% 77% HeLa (human cervix) Lamin A/C % -- GAPDH % -- HeLa-luc (human cervix) Luciferase* 84% 82% Hepa-luc (mouse liver) Luciferase* -- 92% HepG2 (human liver) MAPK1 % -- NIH 3T3-lux (mouse fibroblast) Luciferase* 85% 89% NIH 3T3-L1 MAPK1 7% -- MAPK3 7% -- Secondary Human Astrocytes Lamin A/C % -- ABC A1 7% -- Primary Mouse Hepatocytes Lamin A/C 81% -- PPAR-alpha -- 82% Luciferase Expression (%) non-targeting sirna luciferase non-targeting sirna LDH anti-firefly sirna luciferase anti-firefly sirna LDH 1.5 µl 2.5 µl 3.5 µl.5 µl 1. µl 1.5 µl.5 µl 1. µl 1.5 µl.25 µl 1.25 µl 2.5 µl TransIT-TKO Lipofectamine RNAiMax Lipofectamine DharmaFECT 1 TABLE 2. Knockdown of Genes Using TransIT TKO or TransIT siquest Transfection Reagents. Cells were transfected with sirnas targeting the indicated genes using the TransIT TKO or TransIT siquest Reagents, and the knockdown percentage was determined using quantitative RT-PCR or luciferase assays. *Firefly luciferase expression vectors were stably integrated into the parent cell lines and clonal lines constitutively expressing firefly luciferase were used. FIGURE 27. High Efficiency Knockdown and Low Toxicity Using TransIT-TKO Reagent in HeLa cells. Firefly and sea pansy luciferase reporter vectors were co-transfected into HeLa cells using TransIT -LT1 Reagent. Cells were incubated for at least 4 hours, split into 24-well plates, allowed to adhere and transfected with 25 nm of either a non-targeting sirna or an anti-firefly luciferase sirna using the indicated reagents with the volumes noted beneath each well. Luciferase expression, normalized to non-targeting sirna control (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as percent cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph Toxicity (%) FIGURE 28. High Knockdown and Low Toxicity Using TransIT-siQUEST Reagent in CHO Cells Stably Expressing Firefly Luciferase. CHO-luc cells were grown in 24 wells plates and transfected with 25 nm of either a non-targeting sirna or a anti-firefly luciferase sirna using the indicated reagents with the volumes noted beneath each well. Luciferase expression, normalized to non-targeting sirna control (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as percent cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph. Luciferase Expression (%) non-targeting sirna luciferase non-targeting sirna LDH anti-firefly sirna luciferase anti-firefly sirna LDH.5 µl 1.5 µl 2.5 µl.5 µl 1. µl 1.5 µl.5 µl 1. µl 1.5 µl.25 µl 1.25 µl 2.5 µl TransIT-siQUEST Lipofectamine RNAiMax Lipofectamine DharmaFECT DNA Transfection >> Toxicity (%)

23 Large RNA (Viral RNA and mrna) TransIT -mrna TRANSFECTION KIT High Efficiency Delivery Ensures experimental success by effectively transfecting RNA into a large percentage of the cell population Low Cellular Toxicity Maintain cell density and reduce transfection induced toxicity Serum Compatible Perform transfections in the presence of serum which eliminates the need for a media change and maintains cellular health Deliver Various Sizes of RNA Ideal for specialized applications, such as viral production, protein expression from mrna, and stem cell reprogramming PRODUCT NO. MIR 2225 MIR 225 MIR 2255 MIR 2256 QUANTITY.4 ml 1. ml 5 x 1 ml 1 x 1 ml To inquire about bulk pricing, please call (toll free in the U.S.) or IDEAL FOR USE IN VIRUS PRODUCTION Our lab recently used the TransIT -mrna Transfection Kit to show that intracellular delivery of HPLC-purified and pseudouridine-containing mrna can translate very efficiently without immune activation which is ideal for mrna-based gene therapy applications. TransIT -mrna further facilitated this work through low toxicity transfections of HEK 293T, human dendritic cells (DCs) and primary keratinocytes (Karikó et al. Nucleic Acids Research, 39:e142, 11). Dr. Katalin Kariko, Department of Neurology University of Pennysylvania Description TransIT -mrna Transfection Kit provides high efficiency transfection of large RNA molecules such as mrna or viral RNA. The kit is easy to use and minimizes cellular toxicity due to its ability to transfect RNA in the presence of serum. DNA Transfection 4, EGFP Positive Cells (%) % EGFP Positive Cell Count 3, 2, 1, Cell Count (viability) TransIT -mrna RNAiMax Stemfect 1:1:1 4:1 2:1 FIGURE 29. High Efficiency and Low Toxicity Transfection Following 14 Consecutive Transfections with TransIT -mrna Transfection Kit. Repeated daily transfections were performed in the same population of BJ fibroblasts using three commercially available transfection reagents TransIT-mRNA Transfection Kit (Mirus Bio), Lipofectamine RNAiMAX (Life Technologies) and Stemfect RNA Transfection Kit (Stemgent) - with a capped and polyadenylated EGFP mrna incorporating pseudouridine and 5mC modified bases (Trilink Biotechnologies, Inc.). Multiple reagent-to-rna ratios were tested and the optimal ratio is represented. Transfections were performed in 12-well plates using the indicated reagent-to-rna ratios to deliver 1 µg of RNA. Transfection efficiency was measured by flow cytometry on a guava easycyte 5HT Flow Cytometer following 14 consecutive daily transfections (blue bars). Cell viability was determined using cell counts measured during flow cytometry (black line grey bars). Error bars represent the standard deviation of triplicate wells. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 21

24 TransIT -mrna Transfection Kit continued 1,,, Total Luciferase Activity/Well (RLUs) 1,,, 1,,,,,,,,,,, A549 CHO-K1 COS-7 HEK293 HeLa NIH3T3 Vero FIGURE 3. High Level Luciferase Expression after Delivery of a Luciferase mrna using the TransIT -mrna Transfection Kit. Cells in 12-well plates were transfected with a capped and polyadenylated mrna encoding luciferase using the TransIT-mRNA Transfection Kit. Approximately 18 hours post-transfection the cells were harvested and the total luciferase activity per well was determined. RNA Transfection % GFP Primary Murine BMDC JAWS II Cell Line DC 2.4 Cell Line FIGURE 31. Multiple Dendritic Cell Types Express GFP from mrna Transfected using TransIT -mrna Transfection Kit. Murine primary bone marrow derived dendritic cells (BMDC) and murine dendritic cells types (JAWS II and DC 2.4) were transfected with 1 μg of capped and polyadenlyated mrna encoding GFP using a TransIT-mRNA Reagent: Boost: mrna ratio of 1:1:1 (μl:μl:μg). All cells were seeded (, cell/ well) overnight in 24-well plates. Cells were assayed via flow cytometry 8 hours post transfection. Error bars represent the standard deviation of at least 3 separate experiments. Data courtesy of Kyle Phua (Principal Investigator: Kam W. Leong), Duke University. No RNA Control MHV RNA Transfected FIGURE 32. TransIT -mrna Transfection Kit Successfully Delivers Viral RNAs 32 kb Long. A 32 kb in vitro transcript of the murine coronavirus, MHV, was transfected into DBT cells using the TransIT-mRNA Transfection Kit. Successful transfection assessed by the formation of syncytia hours post-transfection. Syncytia were visualized by phase contrast microscopy. Data courtesy of Mark Clemenz, Loyola University of Chicago. 22 RNA Transfection >>

25 RNAi Controls Label IT RNAi DELIVERY CONTROLS Chemically Labeled Using Mirus established covalent Label IT technology Sensitive Easily visualize transfected cells and assay delivery efficiency using fluorescent microscopy Inert and Compatible Does not target known mammalian genes or cause off-target effects; suitable for co-delivery experiments with functional sirna LABEL PRODUCT NO. QUANTITY Cy 3 MIR 79 1 µg MIR 791 µg Fluorescein MIR µg MIR 793 µg To inquire about bulk pricing, please call (toll free in the U.S.) or Ready-to-Use Supplied as prelabeled sirna control 1 µm solution with 1X RNAi Dilution Buffer Description Label IT RNAi Delivery Controls consist of either Cy 3 or fluorescein labeled sirna duplex that has the same length, charge and configuration as standard sirna. The sequence of the Label IT RNAi duplex is inert and is not known to affect any cellular events. These controls can be co-transfected with a functional target gene-specific sirna and are designed to facilitate assessment of sirna delivery efficiency for in vitro and in vivo applications. Covalent Bond Label Labeled IT RNAi Nucleic Delivery Acid Control RNA Transfection Transfect Label IT RNAi Delivery Control and Detect by Fluorescence Microscopy FIGURE 33. Ready-to-use Label IT RNAi Delivery Controls are Labeled Using the Covalent Label IT Technology. Mirus Label IT reagents* are used to chemically attach labels to a nontargeting sirna at optimized label/base pair ratio to generate Label IT Plasmid Delivery Controls. These pre-labeled Label IT RNAi Delivery Controls facilitate direct tracking of sirna delivery by fluorescence microscopy for in vitro and in vivo studies. The image shows HeLa cells transfected in serum-containing media using Label IT Fluorescein RNAi Delivery Control (green) using the TransIT-TKO Transfection Reagent. Twenty-four hours post-transfection, the cells were fixed, then counterstained to locate the nuclei (blue) and the actin (green). *Label IT kits for a wide variety of applications are also available for purchase. Visit mirusbio.com/labeling for more information. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 23

26 Electroporation Plasmid DNA, RNA, sirna and mirna INGENIO ELECTROPORATION KITS & SOLUTIONS High Efficiency Electroporation Deliver DNA or RNA to hard-to-transfect, stem and primary cells Compatible with Most Conventional Electroporation Devices Use your existing system including Lonza-Amaxa, Bio-Rad, or Harvard BTX Save Money and Reduce Research Costs Without Sacrificing Performance Ingenio Electroporation Solution is available as a stand-alone solution or as part of a complete kit with cuvettes and cell droppers Description Ingenio Electroporation Solution facilitates efficient and reliable delivery of nucleic acids to eukaryotic cells refractory to chemical transfection. Ingenio is a broad spectrum solution that supports high efficiency electroporation with minimal toxicity and replaces standard electroporation solutions including phosphate buffered saline and serumfree media. Ingenio Kits (include solution, cuvettes and cell droppers) are compatible with multiple instruments and facilitate a wide range of applications requiring nucleic acid delivery to cells. It is also available as a stand alone solution. I was very depressed for the last 6 months because I was unable to transfect my rat cell line with various transfection reagents. I tried 5 Nucleofection programs, 2 buffers and several different cell densities. But nothing worked. I am very happy to inform you, Ingenio is a life saver! Sanal Madhusudana Girija, Albert Einstein College of Medicine 24 Ingenio Electroporation Kits for Lonza- Amaxa Nucleofector Devices (solution,.2 cm cuvettes, cell droppers) PRODUCT NO. MIR 5112 MIR 5115 MIR 5118 QUANTITY 25 RXN 5 RXN RXN Ingenio Electroporation Kits for All Other Electroporators, such as Bio-Rad and Harvard-BTX (solution,.4 cm cuvettes, cell droppers) PRODUCT NO. MIR 5113 MIR 5116 MIR 5119 QUANTITY 25 RXN 5 RXN RXN Ingenio Electroporation Solution PRODUCT NO. QUANTITY MIR RXN MIR RXN MIR 5117 RXN Ingenio Electroporation Accessories Cuvettes PRODUCT NO. SIZE QUANTITY MIR 51.2 cm 25 PK MIR cm 5 PK MIR cm 25 PK MIR cm 5 PK Cell Droppers PRODUCT NO. MIR 5124 MIR 5125 QUANTITY 25 PK 5 PK Electroporation >>

27 Ingenio Electroporation Kits and Solutions continued EGFP Positive Cells (%) HL- K562 Jurkat E6-1 SK-N-MC Ingenio Solution using Gene Pulser Xcell TM System Ingenio Solution using Amaxa Nucleofector 2b Device Amaxa Nucleofector Solution V on Amaxa Nucleofector 2b Device FIGURE 34. Ingenio Solution Provides Comparable Efficiency on Amaxa's Nucleofector 2b Device. Cells were electroporated in parallel with an EGFP reporter vector. Two electroporators were used with different electroporation kits: the Ingenio Electroporation Kit was used in the Gene Pulser Xcell Eukaryotic System (Bio-Rad) and the Amaxa Nucleofector 2b Device (Lonza); the Amaxa Nucleofector Kit V was used in the Amaxa Nucleofector 2b Device, all according to manufacturers' recommendations. EGFP expressing cells were identified 24 hours post-electroporation by flow cytometry and presented as a percentage of the live cell population. Experiments were performed in triplicate on three separate days and the data averaged. A. B. FIGURE 35. High Efficiency Plasmid DNA Electroporation of Human Induced Pluripotent Stem (ips) Cells using Ingenio. Ingenio Electroporation Kit was used to transfect 2 x 1 6 ips cells on the Amaxa Nucleofector 2b Device. Cells were electroporated with 8 µg ZsGreen expressing plasmid (Clontech) in µl and plated in 6-well plates at.33 x 1 6 cells/well. Cells were visualized 24 hours post-transfection and imaged under 4X objective with an Olympus IX71 Inverted Microscope. Image is (A) green fluorescence. Cells were also assayed 24 hours post-transfection on an Accuri Cytometer. The histogram (B) shows unelectroporated cells (black line) compared to cells electroporated with plasmid using the Ingenio Electroporation Kit (green line). Data courtesy of Cellular Dynamics International. The Ingenio Electroporation Kit is routinely used in our lab to transfect induced pluripotent stem cells (ipscs) via electroporation and yields extremely high transfection efficiency. The Ingenio Kit is entirely compatible with the amaxa Nucleofector and is also a much more cost effective solution. Elizabeth Dominguez, Cellular Dynamics International (CDI) TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 25 Electroporation

28 Ingenio Electroporation Kits and Solutions continued EGFP Positive Cells (%) *Keratinocyte *Rat Cortical Neuron *MEF BHK-21 CHO-K1 COS-7 HEK-293 HeLa Hepa-SEAP Hep G2 HL- HUVEC Jurkat E6-1 K562 MCF-7 NIH-3T3 Immortalized Keratinocytes PC-12 SK-BR-3 SK-N-MC THP-1 Vero *Primary cell types FIGURE 36. Ingenio Electroporation Kits are Ideal for Electroporation in Many Cell Types Using the Amaxa Nucleofector 2b Device. Cells were assayed at 24 hours post-electroporation by flow cytometry and reported as percentage of live cell population. Visit for ideal pulse conditions. Nomalized Firefly Luciferase Expression (%) GenePulser Nucleofector 2b GenePulser Nucleofector 2b CHO-K1 Jurkat E6-1 GenePulser Nucleofector 2b HeLa Luciferase (GL3) sirna Non-targeting sirna Control Electroporation FIGURE 37. Ingenio Kits Effectively Electroporate sirna in the Amaxa Nucleofector 2b and the GenePulser Electroporators. sirna and plasmid DNA were co-electroporated with the indicated cell lines with the Ingenio electroporation solution in.2 cm cuvettes using either the GenePulser (Bio-Rad) or the Amaxa Nucleofector 2b (Lonza). Plasmid encoding firefly luciferase (1 µg/ml ) was co-electroporated with 25nM of either non-targeting sirna control or GL3 sirna into Jurkat E6-1, HeLa and CHO-K1 cells. Twenty-four hours post electroporation, cells were harvested and assayed for luciferase activity. Data from independent experiments performed on different days were averaged then scaled to non-targeting sirna control and are represented as a percentage of the control. 26 Electroporation >>

29 Ingenio Electroporation Kits and Solutions continued EGFP Positive Cells (%) *HUVEC *Keratinocyte *MEF A549-Flpln BHK-21 CHO-K1 COS-7 HEK-293 HeLa Hep G2 HL- Jurkat E6-1 K562 MCF-7 NIH-3T3 Imortalized Keratinocytes PC-12 RAW264.7 SH-SY5Y SKBR3 SK-N-MC THP-1 U937 Vero *Primary cell types FIGURE 38. Ingenio Electroporation Kits are Ideal for Electroporation in Many Cell Types Using the Bio-Rad GenePulser Xcell System. EGFP expressing cells were identified 24 hours post-electroporation by flow cytometry and presented as a percentage of the live cell population. Visit for ideal pulse conditions. A. B. EGFP Positive Cells (%) HL- K562 Jurkat E6-1 SK-N-MC Propidium Iodide Negative Cells (%) HL- K562 Jurkat E6-1 SK-N-MC Ingenio Electroporation Solution PBS Gene Pulser Electroporation Buffer Ingenio Electroporation Solution PBS Gene Pulser Electroporation Buffer FIGURE 39. Ingenio Kits Outperforms Other Electroporation Solutions in Efficiency and Viability. Cells were electroporated in parallel with an EGFP reporter vector using either Ingenio Electroporation Solution, PBS or GenePulser Electroporation Buffer (Bio-Rad) on the GenePulser Xcell Eukaryotic System. (A) EGFP expressing cells were identified 24 hours post-electroporation by flow cytometry and presented as a percentage of the live cell population. (B) Cells were assayed for viablility by propidium iodide staining and flow cytometry analysis. Error bars represent the standard deviation of triplicate wells. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com 27 Electroporation

30 Terms and Conditions 1. Governing Provisions: All Mirus Bio LLC products are sold and shipped subject to these Terms and Conditions, unless modified by a quotation issued by Mirus Bio LLC or an agreement signed by both Mirus Bio LLC and the customer. These Terms and Conditions supersede any other written document or oral discussion including terms or conditions appearing on a purchase order issued by the customer. 2. Product Safety: Mirus Bio LLC manufactures products for research use by qualified individuals. These products may contain chemicals that may be harmful if misused. Customers are responsible for reviewing and following the instructions included in the product protocols and MSDS that accompany the product. Please contact techsupport@mirusbio. com with any questions regarding the safe use of Mirus Bio LLC products. We recommend our products be used in accordance with the NIH guidelines developed for genetic research. 3. Warranties: Mirus Bio LLC products are warranted to meet or exceed the stated specifications for the usable life of the product defined as either the expiration date for the product or six months from the date of receipt for those products not carrying an expiration date. This warranty is void if the customer has altered or misused the product or failed to store the product as recommended. The warranty does not include defects or failures associated with materials supplied to Mirus Bio LLC by the customer. Mirus Bio LLC disclaims any and all responsibility for any injury or damage which may be caused by the failure of the buyer or any other person to use these products in accordance with the conditions outlined herein. Mirus Bio's sole obligation and the customer's sole remedy are limited to free replacement of products that fail to meet our minimum performance specifications. THIS WARRANTY IS EXCLUSIVE. 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Patents: Mirus Bio's products and processes are covered by one or more patents and are subject to other trade secret and proprietary rights. No transfer or grant of rights under any patent or patents is made or is to be implied by any provision under these Terms and Conditions other than a limited license to use the products for research only. You agree not to infringe upon such rights or to attempt to reverse engineer, reconstruct, synthesize or otherwise modify Mirus Bio products. Certain applications of Mirus Bio products may require licenses from other parties. Determining the existence and scope of such third party intellectual property is the responsibility of the customer. Purchase of the product provides the customer with a limited non-transferable license under any Mirus Bio patents or patent applications to use the product for internal research unless there is a written limitation to this license in the product literature. The customer is responsible for carefully reviewing the product literature and respecting any limitations to this license, e.g. limitations for commercial use or research by for-profit institutions. 5. Customer agrees to indemnify and hold Mirus Bio LLC harmless from and against any and all claims, losses, damages, costs and expenses (including reasonable attorneys fees and amounts paid in settlement in good faith) which may be suffered or incurred by Mirus Bio LLC (including, without limitation, as a result of any claim or action by a third party) arising in any manner from Customer's use of any Products sold from this Web site or otherwise arising out of any act or omission of Customer, its employees, agents or representatives, or by reason of breach of or failure to perform any of Customers obligations hereunder. 6. Orders, Shipment and Delivery: Purchase orders placed before 4: pm Central Time will be shipped that day. Orders placed on Friday will ship on the following Monday for delivery on Tuesday. Orders for regularly stocked items may only be cancelled prior to shipment of the order. Shipping charges are prepaid and added to the customer's invoice. Orders are shipped by Federal Express. Unless agreed upon in writing, all shipments are free carrier (FCA) our facility in Madison, Wisconsin. Costs for transportation and risk of loss transfer to the buyer upon delivery to the carrier. The customer is responsible for any tariffs, duties or taxes associated with international shipments. 7. Copyright and Trademarks: All content contained in printed materials or on the Mirus Bio LLC website is the copyright of Mirus Bio LLC unless specifically identified otherwise. Trademarks referenced herein are either registered trademarks of Mirus Bio LLC or third party trademarks, which are the property of their respective owners. All rights are reserved. Please contact sales@mirusbio.com to request permission to use copyrighted material or trademarks of Mirus Bio LLC. 8. Payment Terms: All sales are net 3 days of invoice in U.S. dollars unless other terms are agreed to in writing by Mirus Bio LLC. Prices are subject to change without notice. Mirus Bio LLC accepts the following credit cards: VISA, MasterCard, American Express and Discover. Mail payments to: Mirus Bio LLC 545 Science Drive Madison, WI USA Attention: Accounts Payable Fax: Returns: Mirus Bio LLC must authorize all product returns. Customers wishing to return a product should contact us at techsupport@mirusbio.com. Product that we confirm damaged, incomplete or do not meet our minimum written specifications will be replaced per our warranty (see above) providing such notice is received from the original purchaser within 3 days of receipt of the product. Customers may contact Mirus Bio LLC to cancel an order after shipment or return a product that meets our minimum written specifications. Mirus Bio LLC must authorize these returns. Products must be unopened and returned in their original, intact condition. The customer must bear the cost of shipping the product back to Mirus Bio LLC under the conditions specified by Mirus Bio LLC. All such returns will be subject to a 25% restocking fee. 1. These Terms and Conditions constitute the entire agreement between Mirus Bio LLC and Customer with respect to Customer use of the Web site and Customer purchase of products hereunder, except as the foregoing (i) may be amended from time to time by Mirus Bio LLC, or (ii) as related to Customer purchase of products, may be superseded by any express conflicting terms or supplemented by any express additional terms in a separate written contract signed by authorized representatives of Mirus Bio LLC and Customer. Trademarks All rights reserved. Mirus Bio LLC. All trademarks are the property of their respective owners. HeLaMONSTER, Ingenio, Label IT, MiraCLEAN, µarray, plive, siquest, TransIT, TransIT-Neural, TransIT-PRO and TransIT-TKO are registered trademarks of Mirus Bio LLC. Tracker and TransIT-X2 are trademarks of Mirus Bio LLC. Reagent Agent is a registered service mark of Mirus Bio LLC. Other Trademarks Alvetex is a registered trademark of Reinnervate Limited. FuGENE is a registered trademark of Fugent LLC. Gene Pulser Xcell is a trademark of Bio-Rad Laboratories, Inc. icell is a registered trademark of Cellular Dynamics International. easycyte is a trademark and guava is a registered trademark of EMD Millipore. Lipofectamine is a registered trademark and NCode is a trademark of Life Technologies Corporation. Amaxa and Nucleofector are registered trademarks of Lonza Group Ltd. TransMessenger is a registered trademark of Qiagen Group. Registered names, trademarks, etc. used in the Mirus Bio website, even when not specifically marked as such, are not to be considered unprotected by law. External links on this web site are provided only for the convenience of Mirus Bio web site visitors. Mirus Bio has no interest in, responsibility for, or control over non-mirus Bio affiliated linked sites. Mirus Bio makes no promises or warranties of any kind, express or implied, including those of merchantability and fitness for a particular purpose, as to content of linked sites. In no event shall Mirus Bio be liable for any damages resulting from use of these links even if Mirus Bio has been informed of the possibility of such liability. Patent and Licensing Information NOTICE TO PURCHASER: LIMITED LICENSE Use of certain Mirus Bio TransIT polyamine transfection reagents are covered by U.S. Patent No. 5,744,335, No. 6,1,784, No. 7,11,995, No. 7,1,367 and patents pending. The use of certain other Mirus Bio transfection products are the subject of one or more of U.S. Patent No. 7,335,59, No. 7,655,468 and/or other pending U.S. patent applications. Mirus Bio Label IT nucleic acid labeling and modifying reagents are covered by U.S. Patent No. 6,262,252, No. 6,593,465, No. 7,49,142, No. 7,326,7 and No. 7,491,538. Cy 3 and Cy 5 products or portions thereof are manufactured under license from Carnegie Mellon University and are covered by U.S. Patent No. 5,268,486. This product is sold to the Buyer with a limited license for Research Use Only and is not for clinical, therapeutic or diagnostic use in humans or animals. This product, or parts from this product, may not be re-packaged or re-sold without written permission from Mirus Bio LLC. The buyer agrees not to infringe upon Mirus patents or to attempt to reverse engineer, reconstruct, synthesize or otherwise modify Mirus Bio products. A license from Mirus Bio LLC is required for commercial application of this product. Use of certain TransIT transfection reagents may be covered by one or more of US patents No.7,25,479, No. 7,662,986, No. 7,666,962, and No. 7,714,75 and corresponding claims outside of the US. This product is sold to the Buyer with a limited license for the sole purpose of the Buyer using the product as a transfection agent for in vitro research or evaluation. This product must not be used in vivo (whether for therapeutic, diagnostic or clinical use) in humans or animals. This product must not be used in relation to any ophthalmic application (including, without limitation, ophthalmic devices, solutions for ophthalmic use, and devices used to incorporate, deliver or regulate the release of drugs for the diagnosis, prophylaxis or treatment of human ophthalmic diseases). This product (or any part) must not be re-packaged or resold or otherwise transferred to any third party without the written permission of Mirus Bio. The Buyer agrees not to infringe upon Mirus Bio s patents or to attempt to reverse engineer, reconstruct, synthesize or otherwise modify this product. A license from Mirus Bio may be required for commercial application of this product, or any use other than in accordance with the foregoing. For further information, contact Mirus Bio LLC, 545 Science Drive, Madison WI license@ mirusbio.com. TheTransfectionExperts.com For Technical Support Tel techsupport@mirusbio.com

31 X2 X2 DNA sirna A Transfection Breakthrough NEW!TransIT-X2 Dynamic Delivery System Achieve superior transfections with an advanced nonliposomal, polymeric system that efficiently delivers both DNA and RNA out of the endosome and into the cytoplasm, overcoming a critical barrier to nucleic acid delivery. The TransIT-X2 Dynamic Delivery System gives researchers: X2 X2 X2 Efficiency superior broad spectrum transfection Delivery simultaneous delivery of plasmid DNA and sirna Technology novel, non-liposomal, polymeric technology Functional Co-delivery of Plasmid DNA and sirna. See page 4 for experimenal details. Providing gene delivery expertise since All rights reserved Mirus Bio LLC. TransIT-X2 is a trademark of Mirus Bio LLC. ADVANCE YOUR TRANSFECTIONS. Request a FREE sample: