1. Products > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma) Altogen Biosystems offers the HeLa-S3 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The HeLa-S3 Transfection Reagent is a proprietary cationic lipids formulation, and it has been developed to provide high transfection efficiency with the HeLa-S3 cell line. This cell line is a good host for research in a wide variety of biomedical research. When cultured in vitro, HeLa-S3 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 HeLa-S3 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. HeLa-S3 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 High Density HeLa-S3 cells Atcc.org Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma) The HeLa S3 is named as such because it is the third close that was propagated and isolated from the original well-known HeLa cell line. The “S” in the name is a tribute to Dr. Sabin. The HeLa cell line was derived in 1955 and has been used in a wide array of scientific research since. As a derivative of the HeLa cell line, the two cell lines share many properties. The HeLa S3 cell line is also considered to be an immortal cell line and has also been under scrutiny in the media in terms of the controversial issue of patient’ rights.

3. HeLa-S3 Transfection Protocol 1. Plate 10,000 - 15,000 HeLa-S3 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 HeLa-S3 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 HeLa-S3 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 > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma)

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 > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma)

5. HeLa-S3 Transfection Kit Product Details A proprietary cationic lipids formulation 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 > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma)

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 the Hela-S3 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 > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma)

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 HeLa-S3 cells. DNA plasmid expressing Cyclophilin B or siRNA targeting Cyclophilin B were transfected into HeLa-S3 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 > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma)

8. Pre-optimized transfection protocol for HeLa-S3 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 HeLa-S3 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 HeLa-S3 cell lines Bio-degradable after endocytosis Used for preclinical research worldwide HeLa-S3 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 > HeLa-S3 Transfection Reagent (Cervical Adenocarcinoma)