1. Altogen Biosystems offers the J774A Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The J774A Transfection Reagent is an advanced lipid based formulation that enhances lipid mediated transfection, and it has been developed to provide high transfection efficiency with J774A cells. This cell line is a good host for transfection and the assessment of cellular functionality. When cultured in vitro, J774A 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 J774A 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 Products > J774A Transfection Reagent (Melanoma Cells, TIB-67)

2. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com J774A Cells www.edoj.org.eg Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > J774A Transfection Reagent (Melanoma Cells, TIB-67) The J774A cell line originates from an adult female mouse (Mus musculus) with reticulum cell sarcoma derived from ascites tissue. The J774A cell line has a population doubling time of approximately 17 hours. The cells are similar to macrophages and are active antibody dependent phagocytosis processes. Research indicates that the J774A cell line can synthesize a significant amount of lysosome. The cell line also continuously synthesizes Interleukin 1 beta (Il1b). This cell line serves as an excellent transfection host for DNA transfection and has applications for research related to biological responses.

3. J774A Transfection Protocol 1. Plate 10,000 - 15,000 J774A 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 J774A 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 J774A 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 > J774A Transfection Reagent (Melanoma Cells, TIB-67)

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 > J774A Transfection Reagent (Melanoma Cells, TIB-67)

5. Data Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > J774A Transfection Reagent (Melanoma Cells, TIB-67) Figure 1. Cyclophilin B silencing efficiency was determined by qRT-PCR in the J774A 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).

6. J774A Transfection Kit Product Details Two component formulation enhances lipid mediated transfection efficiency Transfection protocols provided for transfection of proteins, DNA, mRNA, siRNA, shRNA and microRNA Transfection Enhancer reagent provided with the kit Produce higher level of recombinant protein expression with minimal disruption of normal cell function Generate physiologically relevant data you can trust Effective for plasmid DNA/siRNA co-transfection Easy-to-use transfection protocol with reproducible results Low cytotoxicity 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 > J774A Transfection Reagent (Melanoma Cells, TIB-67)

7. Data Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Figure 2. Protein expression of Cyclophilin B in J774A cells. DNA plasmid expressing Cyclophilin B or siRNA targeting Cyclophilin B were transfected into J774A 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. Products > J774A Transfection Reagent (Melanoma Cells, TIB-67)

8. Pre-optimized transfection protocol for J774A 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 J774A 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 J774A cell lines Bio-degradable after endocytosis Used for preclinical research worldwide J774A 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 > J774A Transfection Reagent (Melanoma Cells, TIB-67)