NIH3T3 Transfection Reagent - Efficient Transfection for Swiss Mouse Fibroblasts

1. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) Altogen Biosystems offers the NIH3T3 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. NIH3T3 Transfection Reagent is a proprietary cationic lipids formulation, and it is developed to provide high transfection efficiency with NIH3T3. This cell line is a good host for studying mouse viruses and human embryonic stem cells. When cultured in vitro, NIH3T3 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 NIH3T3 Transfection Kit at 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

2. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) NIH3T3 Cell Line Description and Applications The NIH3T3 cell line was established in 1962 by researchers at the New York University School of Medicine. It was derived from cells taken from a Swiss mouse embryo. The 3T3 cell line is a type of murine fibroblast cell and is now the classic fibroblast cell line in research. Furthermore, the cell line can be used as “feeder cells” for research related and maintenance of undifferentiated human embryonic stem cells. Murine embryonic fibroblasts, and in particular the 3T3 cell line can be used to secrete growth factors to cultivate keratinocytes. Research indicates that NIH3T3 cells are susceptible to leukemia and sarcoma viruses and that benzodiazepines inhibit the growth of this cell line. The 3T3 cell line is an excellent transfection host and is also useful for titration studies related to mouse viruses. Image of NIH3T3 Cells 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

3. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) NIH3T3 Transfection Protocol 1. Plate 10,000 - 15,000 NIH3T3 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 NIH3T3 cells (from step 2) 7. Incubate cells at 37ºC in a humidified CO2 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 NIH3T3 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

4. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) 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

5. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) NIH3T3 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

6. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) Data Figure 1. GAPD mRNA levels were quantified using real-time RT-PCR in NIH3T3 cells transfected with siRNAs targeting GAPD or non-silencing siRNA. Forty-eight hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for GAPDH mRNA expression levels. Data were normalized against the 18S rRNA signal. Control samples were either mock-transfected or untreated. Values are normalized to untreated sample. Data are means ± SD (n=3).

7. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) Data Figure 2. Protein expression of GAPDH in NIH3T3 cells. DNA plasmid expressing GAPDH or siRNA targeting GAPDH were transfected into NIH3T3 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.

8. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >NIH3T3 Transfection Reagent (Swiss Mouse Fibroblasts) NIH3T3 Transfection Kit Benefits • Pre-optimized transfection protocol for NIH3T3 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 NIH3T3 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 NIH3T3 cell lines • Bio-degradable after endocytosis • Used for preclinical research worldwide Altogen Biosystems 848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone 702 349 6103  Fax 702 989-0841  email  techserv@altogen.com