Description. Lipodin-Pro TM - Protein Transfection Reagent. 1. Kit Benefits

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1 Description Lipodin-Pro TM - Protein Transfection Reagent The delivery of proteins inside living cells represents an alternative to nucleic acids transfection and a powerful strategy for functional studies or therapeutic approaches. Several technologies based on the use of peptide transduction domain (PTD) were developed successfully to transduce proteins across the plasma membrane. However, these PTD poorly interact with proteins, and covalent linkage between the protein and PTD is most often required. Lipodin-Pro is a formulation of lipids able to capture proteins through electrostatic and hydrophobic interactions. There is no need for covalent linkage procedure, Lipodin-Pro is directly mixed with the protein of interest for 10 minutes. The mixture is then added to the cells in culture, the lipid-protein complexes are internalized by the cells and the proteins are released into the cytoplasm within few hours without any cytotoxicity. The optimized formulation of Lipodin-Pro is fully biodegradable maintaining a high cell viability upon delivery. The proteins delivered inside the cells with Lipodin-Pro retain both their structure and function whether peptides, proteins or antibodies are used. 1. Kit Benefits Lipodin-Pro can be used in various functional studies for cell signaling and apoptotic assays, protein-protein interaction, protein localization and compartment shuttling. When the protein is conjugated to a fluorescent dye, the functional assay can be carried out in living cells under multiple treatments with a single sample. Principal Lipodin-Pro advantages: No need for DNA cloning or nucleic acid transfection No chemical ligation or crosslinking Serum compatible, no cytotoxicity and biodegradable Easier 2-step protocol with ready-to-use reagents Deliver functionally active protein within hours Higher delivery efficiency with stable cell lines and primary cells 1

2 2. Transfected Proteins Lipodin-Pro Experimental Results Lipodin-Pro successfully delivered numerous proteins in a wide variety of cells: B and R-phycoerythrin bovine serum albumin β-galactosidase human active caspase-3, caspase 8 and caspase 9 immunoglobulins (unconjugated or labeled with FITC, TRITC, AlexaFluor 488 and AlexaFluor 546) MBP-fusion protein Impurities, contaminants and additives present with the protein of interest affect delivery efficiency. We recommend using a protein sample as pure as possible. We noticed that protein preparations containing high contents of detergents or sodium azide were not compatible with protein transfection, whereas glycerol has no effect. 2

3 3. Cell Types Tested Lipodin-Pro Experimental Results Lipodin-Pro is applicable on numerous cell types. This reagent has been successfully tested on a variety of immortalized cell lines as well as primary cells (Table 1). By submitting end-user data to our Technical Department, we can update this list and improve our technical support to the scientific community. If a particular cell type is not listed, this does not imply that Lipodin-Pro is not working for that type but that it has not yet been tested. Table 1: Example of cells successfully tested with Lipodin-Pro reagent. Cell Line Cell Type Source Efficiency 3T6 Embryonic fibroblasts Mouse 50% A549 Non-small cell lung carcinoma Human 50-80% B16-F10 Melanoma Mouse 50% BEAS-2B Bronchial epithelial cells Human 80% BHK21 Fibroblasts (Kidney) Hamster 80-90% CHO-K1 Epithelial-like (Ovary) Hamster 50-80% COS-1, COS-7 Fibroblasts (Kidney) Green Monkey 50-70% HaCaT Keratinocytes Human 50-80% HEK-293 Transformed Embryonic (Kidney) Human % HeLa Cervical Epithelial Carcinoma Human 50-60% Jurkat T cell leukemia Human 50% L929 Fibrosarcoma Mouse 80-90% K562 Myelogenous leukemia Human 10-50% MDCK Epithelial (Kidney) Canine 10-50% N2A Neuroblastoma Mouse 60-80% NIH3T3 Fibroblasts Mouse 50-75% Raw264.7 Monocytes/macrophages Mouse 90% U87 Glioblastoma Human 50% Vero 10A1 Epithelial (Kidney) Monkey 50% Primary cells Neurons Rat 50% Glial cells Rat 50% 4. Delivery of B and R-Phycoerythrin NIH3T3 A

4 RAW BHK21 B-Phycoerythrin (1 µg, Sigma-Aldrich) was delivered in the indicated cell lines with 2 µl of Lipodin-Pro reagent. Phycoerythrin/Lipodin-Pro complexes were incubated for 24 hours in 24-well plates before unfixed cells were observed by fluorescence microscopy. BEAS-2B BEAS-2B 3T6 HeLa 4

5 Vero Raw R-Phycoerythrin (1 µg, Molecular Probes) was delivered in the indicated cell lines with 2 µl of Lipodin-Pro reagent. Phycoerythrin/Lipodin-Pro complexes were incubated for 24 hours in 24-well plates before unfixed cells were observed by fluorescence microscopy. 5. Delivery of Beta-Galactosidase HeLa CHO-K1 A549 A

6 β-galactosidase (1 µg) was delivered in various cells with 2 µl of Lipodin-Pro reagent. β-galactosidase/lipodin- Pro complexes were incubated for 24 hours in 24-well plates before fixed cells were stained for betagalactosidase activity. 6. Delivery of BSA-TRITC BEAS-2B NIH3T3 Jurkat BHK21 Tetramethylrhodamine-labeled BSA (BSA-TRITC, 2 µg) was delivered in various cells with 3 µl of Lipodin-Pro reagent. BSA-TRITC/Lipodin-Pro complexes were incubated for 24 hours with cells in 24-well plates before unfixed cells were observed with a fluorescent microscope. 6

7 7. Delivery of MBP Fusion Protein MBP-Protein Lipodin-Pro MBP-Protein alone MBP-Protein delivery with the Lipodin-Pro reagent The MBP-fusion protein (10 µg) was delivered with 25 µl of Lipodin-Pro reagent in HEK 293 cells. After an 8-hour incubation, cells were fixed and immunostained with an anti-mbp antibody. Then, the cells were observed by fluorescence microscopy. After a 10-hour incubation, cells were lysed for protein detection by western-blot. 8. Delivery of Immunoglobulins A Nuclear Pore Complex Protein antibody labeled with AlexaFluor 488 (0.5 µg) was mixed with 2 µl of Lipodin-Pro reagent and incubated for 24 hours with BEAS-2B cells in a 24-well plate. Then, cells were fixed with 2% pfa and observed by fluorescence microscopy. A control antibody labeled with AlexaFluor 488 (0.5 µg) was mixed with 2 µl of Lipodin-Pro reagent and incubated for 24 hours with BHK-21 cells in 24-well plates. Then, cells were fixed with 2% pfa and observed by fluorescence microscopy. 7

8 9. Kinetics of R-Phycoerythrin Delivery in NIH3T3 Cells Amount of protein/cell % Fluorescent cells Amount of protein (arbitrary unit) Fluorescent Cells % 0 0 2h 4h 24h R-Phycoerythrin (1 µg) was delivered in NIH3T3 cells with 2 µl of Lipodin-Pro reagent in 24-well plates. Cells were collected and fixed with 2% PFA at the indicated time point. The number of fluorescent cells and the mean fluorescence was determined by cytofluorimetry. The mean fluorescence was used to evaluate the amount of R- Phycoerythrin internalized inside cells. 10. Delivery of Active Caspase-3 Induces Apoptosis HeLa cells in a 24-well plate were treated with 15 ng of active human caspase-3 (Biovision) mixed with 5 µl Lipodin-Pro reagent. Images were taken 6 h after treatment (top row). The bottom image is a control of cells treated with Lipodin-Pro reagent alone. Identical results were obtained with NIH-3T3 cells. W 7

9 An apoptosis assay was performed to quantify apoptotic cells. HeLa cells were treated with 15 ng of active human caspase-3 mixed to 5 µl of Lipodin-Pro reagent in 24 well plates. After a 7-hour incubation, cells were stained with both Annexin-FITC and propidium iodide. Apoptotic and dead cells were counted by cytofluorimetry. A positive control with staurosporine (100 nm) was used to induce apoptosis. Negative controls were cells treated with caspase 3 or Lipodin-Pro alone Staurosporine Caspase-3 + Pro-DeliverIN Lipodin-Pro Caspase-3 Pro-DeliverIN Lipodin-Pro Apoptotic Cells % In another set of experiments, HeLa and A549 cells were treated with 15 ng of active human caspase-3 mixed to 5 µl of Lipodin-Pro reagent in 24 well plates. After a 24-hour incubation, live cells were counted in each well and results plotted as percentage relative to non-treated cells. A positive control with staurosporine (1 µm) was used to induce apoptosis. Negative controls were cells treated with caspase 3 or Lipodin-Pro alone A549 HeLa 20 0 Stau µm 1 µm Caspase-3 r I N Lipodin-Pro N 1 v e ve r I u e l +Lipodin-Pro i eli St a r Pō D Ḏ Pro + sp - 3 Ca Caspase-3 Non-treated p-3 at ed Ca s T re No n 8

10 Overall, Lipodin-Pro reagent can efficiently deliver various types of proteins in different living cell lines. The delivery efficiency is cell type and protein dependent with higher yield for acidic proteins. The delivered protein is still active once inside the cell for binding to target proteins and activating cellular pathways. 9