1. Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells) Altogen Biosystems offers the B16-F10 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. B16-F10 Transfection Reagent is an advanced formulation of a lipid based reagent, and it has been developed to provide high transfection efficiency with B16-F10 and other mouse melanoma cell lines. This cell line is a good host for experimental cellular and molecular biology. When cultured in vitro, B16-F10 cells are a monolayer and adhere to the surface of the culture flask. Other applications include drug discovery, gene expression studies, molecular and cell biology research applications. Purchase B16-F10 Transfection Kit at www.Altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

2. B16-F10 Cell Line Description and Applications Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products The B16-F10 cell originates from the melanoma skin cells derived from a mouse or Mus musculus that was suffering from melanoma. The cell line display a mix of spindle and epithelial morphology. The B16- F10 cell line is extremely weakly immunogenic. The B16-F10 cell line has applications for research in experimental cellular and molecular biology. Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells) High Density B16-F10 Cells atcc.org

3. B16-F10 Transfection Protocol 1. Plate 10,000 - 15,000 B16-F10 cells per well in 0.5 ml of complete growth medium 12–24 hours prior to transfection 2. Wash with 1xPBS and add 0.5 ml of fresh growth medium 3. Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µl of transfection reagent, and 750 ng DNA (or mRNA), or 30 nM - 50 nM of siRNA (or microRNA) *Referred to a final volume including growth medium 4. Incubate transfection complexes at RT for 15 - 30 minutes 5. Optional: Add 2 µl of Complex Condenser. This reagent reduces the size of transfection complex, therefore increasing transfection efficiency; however it may increase cell toxicity 6. Add prepared transfection complexes to 0.5 ml of complete growth medium with B16-F10 cells (from step 2) 7. Incubate cells at 37ºC in a humidified CO 2 incubator 8. Assay for phenotype or target gene expression 48 - 72 hours after transfection Optional: Transfection efficiency can be increased by addition of Transfection Enhancer reagent. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection If the viability of B16-F10 cells being transfected is affected at 16 - 24 hours post-transfection, the level of cytotoxicity can be decreased by changing the growth medium and eliminating redundant exposure of cells to transfectant Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells)

4. General Lipoplex-mediated Transfection Mechanism of Action Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells)

5. B16-F10 Transfection Kit Product Details Two component formulation enhances lipid mediated transfection efficiency Optimized easy-to-use transfection protocol provided for transfection of siRNA, DNA, mRNA, and microRNA Kit includes Transfection Enhancer reagent and recommended transfection protocol High transfection efficacy in the presence of serum Expand your RNAi application with a reagent optimized for delivery of both siRNA and plasmid Reproducible transfection results Works well for standard reverse transfection and high-throughput applications Developed and manufactured by Altogen Biosystems (www.Altogen.com) Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells)

6. Data Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Figure 1. Cyclophilin B silencing efficiency was determined by qRT-PCR in B16-F10 cells transfected by Cyclophilin B siRNA or non-silencing siRNA control following the recommended transfection protocol. Cyclophilin mRNA expression levels were measured 48 hours post-transfection. 18S rRNA levels were used to normalize the Cyclophilin B data. Values are normalized to untreated sample. Data are presented as means ± SD (n=6). Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells)

7. Data Figure 2. Protein expression of Cyclophilin B in B16-F10 cells. DNA plasmid expressing Cyclophilin B or siRNA targeting Cyclophilin B were transfected into B16-F10 cells following Altogen Biosystems transfection protocol. At 72 hours post-transfection the cells were analyzed by Western Blot for protein expression levels (normalized by total protein, 10 µg of total protein loaded per each well). Untreated cells used as a negative control. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells)

8. Pre-optimized transfection protocol for B16-F10 cell line Compatible with DNA, small RNA (siRNA, shRNA, miRNA), mRNA, and small protein complexing Free of serum and protein of animal origin Compatible with standard and reverse transfection methods (both protocols provided in the kit manual) Easy to use B16-F10 transfection protocol ensures great performance with expedited experimental timeline Equally efficient for single or multiple transfections Can be used for transient transfection and development of stable B16-F10 cell lines Bio-degradable after endocytosis Used for preclinical research worldwide B16-F10 Transfection Kit Benefits Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > B16-F10 Transfection Reagent (Mouse Melanoma Cells)