1. Products > SKNAS Transfection Reagent (Neuroblastoma Cells) Altogen Biosystems offers the SKNAS Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The SKNAS Transfection Reagent is a proprietary cationic lipids formulation, and it has been developed to provide high transfection efficiency with the SKNAS cell line. This cell line is a good host for studying brain cancer and cell and molecular biology. When cultured in vitro, SKNAS 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 SKNAS 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. SKNAS 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 SKNAS cells Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > SKNAS Transfection Reagent (Neuroblastoma Cells) The SKNAS cell line originates from a bone marrow metastasis from a 6-year old Caucasian female child. The patient had poorly differentiated embryonal neuroblastoma. The SKNAS cell line has a modal number of 47 and hyperdiploid. These cells are unique in their production of large amounts of IGF-2 and type 1 IGF receptors. The Memorial Sloan-Kettering Cancer Center only distributes the cell line for research purposes. The SKNAS cell line is particular useful for studying therapeutics related to specific types of brain cancer.

3. SKNAS Transfection Protocol 1. Plate 10,000 - 15,000 SKNAS 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 SKNAS 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 SKNAS 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 > SKNAS Transfection Reagent (Neuroblastoma Cells)

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 > SKNAS Transfection Reagent (Neuroblastoma Cells)

5. SKNAS Transfection Kit Product Details 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 > SKNAS Transfection Reagent (Neuroblastoma Cells)

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 SKNAS 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=3). Products > SKNAS Transfection Reagent (Neuroblastoma Cells)

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 SKNAS cells. DNA plasmid expressing Cyclophilin B or siRNA targeting Cyclophilin B were transfected into SKNAS 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 > SKNAS Transfection Reagent (Neuroblastoma Cells)

8. Pre-optimized transfection protocol for SKNAS 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 SKNAS 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 SKNAS cell lines Bio-degradable after endocytosis Used for preclinical research worldwide SKNAS 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 > SKNAS Transfection Reagent (Neuroblastoma Cells)