1. Products > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739) Altogen Biosystems offers the AGS Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. AGS Transfection Reagent is an advanced formulation of a polymer based reagent, and it is developed to provide high transfection efficiency with AGS and other gastric adenocarcinoma cell lines. This cell line is a good host for studying stomach cancer and human infections related to the stomach. Other applications include drug discovery, gene expression studies, molecular and cell biology research applications. Purchase AGS 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. The AGS cell line is derived from the stomach tissue of a Caucasian female who had adenocarcinoma or stomach cancer, aged 54 years from 1979. No therapy was administered to the patient before the cells were taken. The AGS cell line is tumorigenic and shows epithelial cellular morphology and this line has the potential to be used in research related to different types of stomach cancer. AGS 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 AGS Cell Line Atcc.org Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739)

3. AGS Transfection Protocol 1.Plate 10,000 - 15,000 AGS 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 AGS 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 AGS 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 > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739)

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 > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739)

5. AGS Transfection Kit Product Details Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com 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) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739)

6. Data Figure 1. GAPD mRNA levels were quantified using real-time RT-PCR in the AGS 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 GAPD 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=5). Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739)

7. Data Figure 2. Protein expression of GAPDH in AGS cells. DNA plasmid expressing GAPDH or siRNA targeting GAPDH were transfected into AGS 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 > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739)

8. Pre-optimized transfection protocol for AGS 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 AGS 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 AGS cell lines Bio-degradable after endocytosis Used for preclinical research worldwide AGS 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 > AGS Transfection Reagent (Gastric Adenocarcinoma, CRL1739)