Leading Developer and Manufacturer of In Vivo and

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > HCT-116 Transfection Reagent (Colon Carcinoma) Altogen Biosystems offers the HCT-116 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The HCT-116 Transfection Reagent is an advanced formulation of a biodegradable polymer-based reagent, and it has been developed to provide high transfection efficiency with the HCT-116 cell line. This cell line is a good host for studying kidney cell function. When cultured in vitro, HCT-116 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 HCT-116 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

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > HCT-116 Transfection Reagent (Colon Carcinoma) HCT-116 Cell Line Description and Applications The HCT-116 cell line originates from epithelial tumorigenic tissue taken from the colon of an adult male with a colorectal carcinoma. The cells are positive for transforming growth factor and keratin by immunoperoxidase. The HCT-116 cell line has a mutation in codon 13 of the ras proto-oncogene and as a result, the cell line has applications for PCR studies related to the colon. This cell line is used to study therapeutics associated with colon cancer. High Density HCT-116 cells www. atcc. org 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 > HCT-116 Transfection Reagent (Colon Carcinoma) HCT-116 Transfection Protocol 1. Plate 10, 000 - 15, 000 HCT-116 cells per well in 0. 5 ml of complete growth medium 12– 24 hours prior to transfection 2. Wash with 1 x. PBS 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 m. RNA), or • 30 n. M - 50 n. M of si. RNA (or micro. RNA) *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 HCT-116 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 HCT-116 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 > HCT-116 Transfection Reagent (Colon Carcinoma) 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 > HCT-116 Transfection Reagent (Colon Carcinoma) HCT-116 Transfection Kit Product Details • Proprietary cationic lipids formulation • Optimized for intracellular delivery of plasmid DNA, si. RNA, micro. RNA, and m. RNA • High transfection efficiency of both si. RNA and plasmid DNA without compromising cell viability • Achieve robust si. RNA uptake for dependable gene silencing • Effective transfection under conditions of up to 40% serum • Transfection kit includes Transfection Enhancer reagent • Gentle enough to be used for single cell analysis 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 > HCT-116 Data Transfection Reagent (Colon Carcinoma) Figure 1. si. RNAs targeting Lamin A/C m. RNA or non-silencing control si. RNA were transfected into HCT 116 cells following the recommended protocol. At 48 hours post-transfection the cells were analyzed by q. RT-PCR for Lamin A/C gene expression levels. 18 S r. RNA levels were used to normalize the Lamin A/C data. Values are normalized to untreated sample. Data are means ± SD (n=4).

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > HCT-116 Data Transfection Reagent (Colon Carcinoma) Figure 2. Protein expression of Lamin A/C in HCT-116 cells. DNA plasmid expressing Lamin A/C or si. RNA targeting Lamin A/C were transfected into HCT-116 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 > HCT-116 Transfection Reagent (Colon Carcinoma) HCT-116 Transfection Kit Benefits • Pre-optimized transfection protocol for HCT-116 cell line • Compatible with DNA, small RNA (si. RNA, sh. RNA, mi. RNA), m. RNA, 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 HCT-116 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 HCT-116 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