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Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) Altogen Biosystems offers the ASMC Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. ASMC Transfection Reagent is an advanced formulation of a lipid-based reagent, and it has been developed to provide high transfection efficiency with ASMC and other vascular cell lines. This cell line is an excellent host for studying human vascular disease. When cultured in vitro, ASMC 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 ASMC 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 > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) ASMC Cell Line Description and Applications High Density ASMC Cell Line atcc. org The ASMC or Aortic Smooth Muscle Cells originate from the human tunica intima and tunica media of healthy aorta tissue. The tunica media is made up of mostly smooth muscle cells as well as elastic fibers that are arranged in spirals; it is the middle layer of the artery. The tunica intima is the inner layer of the artery, and it is made up of a layer of elastic fibers and a network of connective tissue. ASMC cells can synthesize glycosaminoglycan, myosin, collagen, and elastin and can also differentiate in response to various factors. The ASMC cells can be used to study atherosclerosis, angiogenesis, stroke, diabetes, vascular biology, diabetes and other human vascular disorders. ASMC cells are sensitive to IGF 1 and IGF 2, but resistant to insulin at the receptor level. Via the inhibition of MMP-9 expression, curcumin can prevent migration of ASMC cells. 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 > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) ASMC Transfection Protocol 1. Plate 10, 000 - 15, 000 ASMC 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 ASMC 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 ASMC 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 > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) 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

Products > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) ASMC Transfection Kit Product Details • Two-component formulation enhances lipid-mediated transfection efficiency • Optimized easy-to-use transfection protocol provided for transfection of si. RNA, DNA, m. RNA, and micro. RNA • 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 si. RNA 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

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) Data Figure 1. Si. RNAs targeting Lamin A/C m. RNA or non-silencing control si. RNA were transfected following the recommended protocol. At 48 hours post-transfection the ASMC cells were analyzed by q. RT-PCR for 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=3).

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) Data Figure 2. Protein expression of GAPDH in ASMC cells. DNA plasmid expressing GAPDH or si. RNA targeting GAPDH were transfected into ASMC 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 > ASMC Transfection Reagent (Aortic Smooth Muscle Cells) ASMC Transfection Kit Benefits • Pre-optimized transfection protocol for ASMC 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 ASMC 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 ASMC 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