YEARS TIO N Transfection Products

Transcription

1 VISION YEARS TIO N 2 RA PASSION ting Celebra INSP I 215 Transfection Products

2 ONE FOCUS: TRANSFECTION Transfection is the introduction of any nucleic acid molecule by non-viral means into cultured eukaryotic cells. This brochure highlights 2 years of intensive scientific discovery and development facilitated by a team of chemists and biologists to deliver the very best transfection reagents in the world. Transfection continues to be the focus and passion of Mirus Bio, providing researchers with products and technical expertise they need to excel. MOST RECENT TRANSFECTION BREAKTHROUGHS 214: TransIT -Insect Effective transient transfection for high yield baculovirus titers 213: TransIT-X2 Dynamic Delivery System Superior delivery of plasmid DNA and/or sirna TransIT -BrCa Transfection Reagent The first breast cancer cell transfection reagent 21: TransIT-PRO Transfection Kit Large-scale, high yield protein production 29: TransIT -22 Transfection Reagent Broad spectrum DNA transfection 28: Ingenio Electroporation Kits & Solution Versatile, multi-platform electroporation solution

3 CONTENTS DNA/RNA TRANSFECTION BROAD SPECTRUM DNA & sirna/mirna TransIT-X2 Dynamic Delivery System DNA TRANSFECTION BROAD SPECTRUM DNA TransIT -22 Transfection Reagent TransIT -LT1 Transfection Reagent CELL LINE SPECIFIC TransIT -293 Transfection Reagent... 1 TransIT -BrCa Transfection Reagent... 1 TransIT -CHO Transfection Kit... 1 TransIT-HeLaMONSTER Transfection Kit... 1 NEW! TransIT -Insect Transfection Reagent TransIT -Jurkat Transfection Reagent TransIT -Keratinocyte Transfection Reagent TransIT-Neural Transfection Reagent INSECT CELL TRANSFECTION AND BACULOVIRUS PRODUCTION NEW! TransIT -Insect Transfection Reagent 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

4 DNA/siRNA Transfection Broad Spectrum DNA & sirna/mirna 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 CAT. NO. QUANTITY ml ml ml x 1.5 ml x 1.5 ml We recently tested the TransIT-X2 Dynamic Delivery System head-to-head against Lipofectamine 2 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 2. 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. FIGURE 1. TransIT-X2 Dynamic Delivery System Enables Superior Gene Expression in a Variety of Cell Types. TransIT-X2 Dynamic Delivery System and Lipofectamine 2 Transfection Reagent were used to transfect plasmid DNA encoding luciferase into 41 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 36 of 41 cell types; 17 cell types that had expression levels 2-fold higher are denoted with. 2 TransIT-X2 Dynamic Delivery System A549 AsPC-1 BHK-21 BJ BT-2 Caco-2 C2C12 CFPAC-1 CHO-K1 DI-TNC1 DU-145 Hep G2 HCC1143 HCC38 HEK 293 HMEC (epithelial) HUVEC Immortalized Keratinocytes FreeStyle TM 293-F MDA-MB-468 MDCK NHDF NIH-3T3 L929 LNCaP PC-12 RAW T47D 28 AU565 BxPC-3 COS-7 HeLa HCT 116 MCF-7 MDA-MB-231 PC Lipofectamine 2 Keratinocytes MDA-MB-453 Neuro-2a SH-SY5Y SK-N-MC Cell types with >2-fold luciferase expression in head-to-head comparisons. DNA/siRNA Transfection >>

5 DNA/siRNA Transfection TransIT-X2 Dynamic Delivery System continued A549 CHO-K1 LNCaP PC-12 Hep G2 MDCK 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 S1 inverted fluorescence microscope. *Indicates primary cell types. 1 9 EGFP Positive (%) A549 CHO-K1 Hep G2 MDCK LNCaP PC-12 HMEC* NHDF* 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. 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 (Cy 5 labeled DNA), red (Cy 3 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 2 GAPDH TransIT-X2 Lipofectamine 2 aha1 DNA/siRNA Transfection FIGURE 5. TransIT-X2 Dynamic Delivery System Achieves Higher Knockdown than Lipofectamine 2. Human TransIT-X2 Dynamic Delivery System and Lipofectamine 2 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 2 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. 1 Knockdown of PTK9 mrna (%) Transfection Reagent mirna TransIT-X2 Lipofectamine 2 Pre-miR hsa-mir-1 TransIT-X2 Lipofectamine 2 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 2 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 2 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. 5

8 DNA Transfection Broad Spectrum DNA TransIT -22 TRANSFECTION REAGENT Broad Spectrum DNA Delivery Achieve high expression in many cell types, including hardto-transfect and primary cells Outperforms Competitor Reagents TransIT -22 demonstrates higher protein yield and less toxicity when compared to other transfection reagents Animal Origin Free provides high performance with maximum compatibility CAT. NO. QUANTITY ml ml x 1. ml x 1. ml IDEAL FOR USE IN VIRUS PRODUCTION Using TransIT -22, we transfected HeLa cells in 6-well plates with 1.25 µg of the Zheng lab construct (px33) from Addgene that harbors both a specific guide RNA against a recognition sequence in our gene of choice, and 1.25 µg of a donor plasmid with 1 kb of 5 and 3 homology sequence. We then selected the cells using puromycin and came across a population that harbored the modification we were interested in. Thank you so much for the sample of TransIT -22. Mirus has always been without exception the gold standard for me and why anyone else would want to use anything else is just beyond me. Aviva Joseph, University of Massachusetts Medical School Description TransIT-22 Reagent is a versatile transfection solution for broad spectrum DNA delivery into mammalian cells. This reagent is animal component free allowing maximum compatibility for all downstream applications while outperforming major competitors in most cell types. FIGURE 7. High Performance Plasmid Transfection. Primary Human Small Epithelial Cells (HSAEpic) were transfected using TransIT -22 and an EGFP expression plasmid (4:1 reagent-to-dna ratio). Images were taken 24 hours posttransfection using a Zeiss Axiovert inverted fluorescence microscope. 6 I recently tested TransIT -22 and TransIT -LT1, and both reagents worked well in terms of their efficiency at transfecting human-derived ips cells with CRIPSR constructs and a fluorescent protein reporter. Through visual inspection, transfection efficiencies with TransIT -22 and TransIT -LT1 were clearly higher than with Lipofectamine 3. Fedir Kiskin, University of Cambridge DNA Transfection >>

9 TransIT -22 Transfection Reagent continued 1 Normalized Luciferase Expression (%) HUVEC THP-1 NT2 CHO-K1 HEK-293 TransIT -22 FuGENE HD Lipofectamine 2 FIGURE 8. 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 -22 (Mirus Bio, 2:1 or 3:1), FuGENE HD (Promega, 3.5:1), Lipofectamine 2 (Life Technologies, 1.5:1, 3:1 or 5:1). Luciferase activity was measured 24 hours posttransfection. Values were normalized to TransIT -22 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 9, 7 8, 6 Luciferase (RLU) 7, 6, 5, 4, Toxicity (%) 3, 2, 2 1, 1 2:1 3:1 4:1 1.5:1 3:1 5:1 1.5:1:1 3:1:1 5:1:1 TransIT -22 Lipofectamine 2 Lipofectamine LTX FIGURE 9. TransIT -22 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. 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 CAT. NO. QUANTITY ml ml x 1. ml x 1. ml IDEAL FOR USE IN VIRUS PRODUCTION 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 212). 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. Luciferase (RLU) 2,5, 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 2 Lipofectamine LTX and PLUS 1.5:1 2:5:1 3 5:1 FuGENE HD FIGURE 1. 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 11. 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. Luciferase Activity (RLU) 1.E E E + 6 DNA Transfection 1.E + 5 CHO-K1 COS-7 HEK 293 HeLa TransIT -LT1 FuGENE 6 FIGURE 12. 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. FIGURE 13. Exceptional Transfection Efficiency in Human Induced Pluripotent Stem Cells (ipscs) via Reverse Transfection with TransIT -LT1 Transfection Reagent. TransIT -LT1 Transfection Reagent was used to reverse transfect 1.3 x 1 6 ips cells with a ZsGreen expressing plasmid (Clontech). Reverse transfections were performed in 6-well plates using 12 µl of TransIT -LT1 Transfection Reagent to deliver 4 µg of DNA (3:1, reagent: DNA). Cells were visualized 48 hours post-transfection and imaged under a 1X objective with an Olympus IX71 Inverted Microscope. The image is green fluorescence. Cells were assayed 48 hours post-transfection on an Accuri Cytometer. Data courtesy of Cellular Dynamics International. 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, and related TransIT -BrCa Transfection Reagent MCF-7, MDA-MB-231, MDA- MB-453, MDA-MB-468, T47D Efficiency** Cat. No. Quantity IDEAL FOR USE IN VIRUS PRODUCTION 75 85% 4 8% ml ml x 1. ml x 1. ml ml ml x 1. ml x 1. ml TransIT -CHO Transfection Kit (TransIT -CHO Reagent & CHO Mojo Reagent) ml ml CHO-K1 and related 5 6% x 1. ml x 1. ml TransIT-HeLaMONSTER Transfection Kit (TransIT -HeLa Reagent and MONSTER Reagent) ml ml HeLa and related 5 6% x 1. ml x 1. ml 1 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 DNA Transfection >>

13 TransIT Cell Line Specific Transfection Reagents continued Product* Applicable Cell Line(s) or Cell Type(s) Efficiency** Cat. No. Quantity NEW!TransIT -Insect Transfection Reagent TransIT -Jurkat Transfection Reagent High Five, S2, Sf9 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 2 3% C6, Daoy, DB-TRG-5MG, DI-TNC1, HCN-1A, Neuro-2a, PC-12, SK-N-MC, SVGp12 >75% ml ml x 1. ml x 1. ml ml ml x 1. ml x 1. ml ml ml x 1. ml x 1. ml ml ml x 1. ml 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. TransIT -CHO Transfection Kit is a great product. Easy to use, works well, and reasonably priced. Matthew Nicotra, University of Pittsburgh 11

14 Insect Cell Transfection and Baculovirus Production NEW! TransIT -INSECT TRANSFECTION REAGENT DNA Transfection Expectional DNA Delivery In insect cell types including Sf9, High Five and S2 CAT. NO. QUANTITY ml High Baculovirus Production Ideal for baculovirus expression in insect cells ml Serum Compatibility Non-liposomal, animal-origin free formulation that eliminates media change x 1. ml x 1. ml Better Value Low reagent amounts required per transfection IDEAL FOR USE IN VIRUS PRODUCTION Description Insect cell expression is a platform used to produce proteins with simple posttranslational modifications. Transient transfection and recombinant baculovirus production are commonly used methods for insect cell expression. TransIT -Insect Transfection Reagent is an animal-origin free transfection reagent specifically optimized for high gene expression in a variety of insect cell types A. B. FIGURE 14. Efficienct Transfection of Baculovirus Genomic DNA Using TransIT -Insect Reagent. Transfections were performed in 6-well plates with 5 x 15 Sf9 cells per well using TransIT -Insect Transfection Reagent at the reagent-tototal DNA ratio of 3:1 (µl:µg). Cells were co-transfected with.5 µg of ProGreen baculovirus genomic vector DNA (AB Vector) encoding green-fluorescent protein (GFP) and.1 µg of pvl1393 transfer vector (AB Vector). (A) Fluorescence and phase contrast images were taken at 6 days post-transfection using a Zeiss S1 fluorescent microscope. Merge shown in (B). FIGURE 15. Superior Recombinant Protein Expression in High Five Cells Using TransIT -Insect. High Five cells were transfected in 6-well plates with 2.5 µg of a GFP expression plasmid driven by an hr5 enhancer/ie1 promoter using the designated reagent at the indicated reagent-to-dna ratios (µl:µg). Total soluble cell lysates were prepared from cells 72 hours post-transfection. Lysates from 1 µl culture were analyzed by SDS-PAGE and Coomassie blue staining; cells alone (untransfected) is shown as control. Expressed GFP containing 6X His, S, and HSV tags (~38 kda) was clearly detected in the lysate from the cells that were transfected (*) with the highest level of expression observed at TransIT -Insect:DNA ratio of 2:1. 12 DNA Transfection >>

15 TransIT -Insect Transfection Reagent continued 14,,, 3,,, Sf9 12,,, 25,,, High Five 1,,, 2,,, Luciferase (RLU) 8,,, 6,,, 4,,, Luc ferase (RLU) 15,,, 1,,, 2,,, 5,,, TransIT Insect Cel fectin II ESCORT IV TransIT Insect Cellfectin II ESCORT IV Luciferase (RLU) 6,,, 5,,, 4,,, 3,,, 2,,, 1,,, TransIT Insect Cel fectin II ESCORT IV FIGURE 16. TransIT -Insect Outperforms Competitor Transfection Reagents. Insect cell lines Sf9, High Five, and Drosophila S2 cells were transfected in 96-well plates with.1 µg of a luciferase expression plasmid driven by an hr5 enhancer/ie1 promoter using the designated reagent at the indicated reagent-to-dna ratios (µl: µg). Luciferase expression was measured at 48 hours post-transfection. Sf9 and High Five cells were cultured and transfected in serum-free media formulations; S2 cells were in serum containing medium. Error bars represent the standard error of the mean for triplicate wells. S2 DNA Transfection 35,,, 3,,, 24 hr 48 hr 72 hr 25,,, Luciferase (RLU) 2,,, 15,,, 1,,, 5,,, Sf9 High Five S2 FIGURE 17. TransIT -Insect Yields Increased Protein Expression Over Time. Insect cell lines Sf9, High Five, and Drosophila S2 were transfected in a 96-well plate with.1 ug of a luciferase expression plasmid driven by an hr5 enhancer/ie1 promoter using the TransIT -Insect Transfection Reagent at a reagent-to-dna ratio of 2:1 (µl: µg). Luciferase expression was measured at three time points, 24, 48 and 72 hours post-transfection. Sf9 and High Five cells were cultured and transfected in serum-free media formulations; S2 cells were in serum containing medium. Error bars represent the standard error of the mean for triplicate wells. Our lab successfully tested TransIT -Insect Transfection Reagent for generating recombinant baculovirus in insect cells. Using TransIT -Insect with multiple BEVS we were able to generate high-titer baculovirus that resulted in consistently higher protein expression in High Five and Sf9 cells compared to Cellfectin II (Life Technologies). (Kuo et al, Protein Eng Des Sel. Oct 212). Dr. Linda Lua (Director), Protein Expression Facility The University of Queensland 13

16 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 CAT. NO. QUANTITY ml ml DNA Transfection Total Cost Savings Higher protein yield translates to lower material and labor costs 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 212). 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. TransIT-PRO outperforms linear PEI in protein yield, while providing a cost-effective alternative to FreeStyle MAX. A. Human Fc (µg/l) 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 2 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) TransIT-PRO + PRO Boost Reagent 25kDa Linear PEI BD Select TM CD1 Medium BD Select TM CHO Medium FreeStyle TM CHO Expression Medium PowerCHO 2 CD Medium ProCHO TM 5 Medium FreeStyle TM Max 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. DNA Transfection 12 1 Yield (mg/l) Protein TransIT-PRO Transfection Reagent (Mirus Bio) 293Fectin (Life Technologies) FIGURE 2. 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. 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 CAT. NO. QUANTITY ml ml x 1. ml x 1. ml 3D Transfection System Includes:.4 ml TransIT-3D Transfection Reagent and 2 alvetex 3D 12-well Plates each 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. 1,5,, 1,25,, 1,,, Luciferase Activity (RLUs) 75,, 3,, 25,, 2,, 15,, 1,, 5,, Reagent-to-DNA Ratio 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 CAT. NO. QUANTITY ml ml x 1. ml x 1. ml 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 CAT. NO. QUANTITY ml ml x 1. ml x 1. ml 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. 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 CAT. NO. QUANTITY Cy µg µg Fluorescein µg µg *Label IT kits for a wide variety of applications are also available for purchase. 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 Transfect Labeled Plasmid and Detect by Fluorescence Microscopy 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). 18 DNA & RNA 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 We have tried other transfection reagents, but only the TransIT-TKO reagent gives us a 1% transfection rate and gene knockdown without toxicity in these cells (RAW 264.7). Nature Protocols, 1: (26) TransIT-TKO Transfection Reagent CAT. NO. QUANTITY ml ml x 1.5 ml x 1.5 ml TransIT-siQUEST Transfection Reagent CAT. NO. QUANTITY ml ml x 1.5 ml x 1.5 ml 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 (%) TransIT-siQUEST Reagent TransIT-TKO Reagent Lipofectamine 2 RNA Transfection Reagent Alone CHO-K1 COS-7 HEK 293 HepG2 MCF-7 NIH 3T3 RAW264.7 FIGURE 26. Knockdown Efficiencies Using TransIT-siQUEST, TransIT-TKO Reagents and Lipofectamine 2. 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 2 (gray) Reagents. Bars indicate the percent of normalized firefly luciferase expression as compared to each reagent alone control 24 hours post-transfection. Vero 19

22 TransIT-TKO & TransIT-siQUEST Transfection Reagents continued Cell Line (Source) Endogenous Transcript TransIT-TKO Knockdown Efficiency TransIT-siQUEST Knockdown Efficiency A549-luc (human lung) Luciferase* 77% 82% BNL CL.2 MAPK1 8% -- (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 8% -- GAPDH 8% -- HeLa-luc (human cervix) Luciferase* 84% 82% Hepa-luc (mouse liver) Luciferase* -- 92% HepG2 (human liver) MAPK1 8% -- NIH 3T3-lux (mouse fibroblast) Luciferase* 85% 89% NIH 3T3-L1 MAPK1 7% -- MAPK3 7% -- Secondary Human Astrocytes Lamin A/C 8% -- ABC A1 7% -- Primary Mouse Hepatocytes Lamin A/C 81% -- PPAR-alpha -- 82% TABLE 1. 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. RNA Transfection Luciferase Expression (%) non-targeting sirna lucife ase non-targeting sirna LDH anti-firefly s RNA luciferase anti-firefly s RNA 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 2 DharmaFECT 1 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. Luciferase Expression (%) non-targeting s RNA 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 2 DharmaFECT Toxicity (%) 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. 2 RNA Transfection >>

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 CAT. NO. QUANTITY ml ml x 1 ml x 1 ml Deliver Various Sizes of RNA Ideal for specialized applications, such as viral production, protein expression from mrna, and stem cell reprogramming 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, 211). Dr. Katalin Karikó, 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. EGFP Positive Cells (%) % EGFP Positive Cell Count IDEAL FOR USE IN VIRUS PRODUCTION 4, 3, 2, 1, Cell Count (viability) RNA Transfection 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. 21

24 TransIT -mrna Transfection Kit continued 1,4,, 1,2,, Total Luciferase Activity/Well (RLU) 1,,, 8,, 6,, 4,, 2,, 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 posttransfection 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 (8, 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 CAT. NO. QUANTITY Cy µg µg Fluorescein µg µg 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 IT RNAi Delivery 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. 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 Amaxa Nucleofector II/2b Nucleofector Devices (solution,.2 cm cuvettes, cell droppers) CAT. NO. QUANTITY RXN RXN RXN Ingenio Electroporation Kits for All Other Electroporators, such as Bio-Rad and Harvard BTX (solution,.4 cm cuvettes, cell droppers) CAT. NO. QUANTITY RXN RXN RXN Ingenio Electroporation Solution CAT. NO. QUANTITY RXN RXN RXN Ingenio Electroporation Accessories Cuvettes CAT. NO. SIZE QUANTITY cm 25 PK cm 5 PK cm 25 PK cm 5 PK Cell Droppers CAT. NO. QUANTITY PK PK Electroporation >>

27 Ingenio Electroporation Kits and Solutions continued 1 EGFP Positive Cells (%) HL-6 K562 Jurkat E6-1 SK-N-MC Ingenio Solution using Gene Pulser Xcell M 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 the Amaxa Nucleofector II/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 II/2b Device (Lonza); the Amaxa Nucleofector Kit V was used in the Amaxa Nucleofector II/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 II/2b Device. Cells were electroporated with 8 µg ZsGreen expressing plasmid (Clontech) in 1 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 II/2b and is also a much more cost effective solution. Elizabeth Dominguez, Cellular Dynamics International (CDI) 25 Electroporation