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 > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) Altogen Biosystems offers the Neuro-2 a Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The Neuro-2 a Transfection Reagent is a liposome formulation reagent, and it has been developed to provide high transfection efficiency with the Neuro-2 a cell line. This cell line is a good host for a wide variety of research in cell and molecular biology. When cultured in vitro, Neuro-2 a 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 Neuro-2 a 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 > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) Neuro-2 a Cell Line Description and Applications The Neuro-2 a cell line originates from brain tissue from an albino house mouse (Mus musculus) with neuroblastoma. The Neuro-2 a cell line displays amoeboid and neuronal stem cell morphology. The cell line produces large amounts of microtubular protein. Research indicates this protein is critical to a nerve cell contractile system that supports axoplasmic flow. The Neuro-2 a cell line is susceptible to several viruses: human poliovirus 1, vesicular stomatitis virus, and herpes simplex virus. This cell line makes an excellent transfection host, and its morphology has different applications in biological research. High Density Neuro-2 a Cells Atcc. 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 > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) Neuro-2 a Transfection Protocol 1. Plate 10, 000 - 15, 000 Neuro-2 a 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 Neuro-2 a 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 Neuro-2 a 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 > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) 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 > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) Neuro-2 a Transfection Kit Product Details • Proprietary cationic lipids formulation • Transfection protocols provided for transfection of proteins, DNA, m. RNA, si. RNA, sh. RNA and micro. RNA • 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/si. RNA 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 > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) Data Figure 1. Cyclophilin B silencing efficiency was determined by q. RT-PCR in Neuro-2 a cells transfected by Cyclophilin B si. RNA or non-silencing si. RNA control following the recommended transfection protocol. Cyclophilin m. RNA expression levels were measured 48 hours post-transfection. 18 S r. RNA levels were used to normalize the Cyclophilin B data. Values are normalized to untreated sample. Data are presented as means ± SD (n=3).

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) Data Figure 2. Protein expression of Cyclophilin B in Neuro-2 a cells. DNA plasmid expressing Cyclophilin B or si. RNA targeting Cyclophilin B were transfected into Neuro-2 a 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

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products > Neuro-2 a Transfection Reagent (Neuroblastoma Cells, CCL 131) Neuro-2 a Transfection Kit Benefits • Pre-optimized transfection protocol for Neuro-2 a 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 Neuro-2 a 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 Neuro-2 a 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