1. Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Altogen Biosystems offers the Astrocyte Cell Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The Astrocyte Cell Transfection Reagent is a proprietary cationic lipids formulation, and it has been developed to provide high transfection efficiency with Astrocytes. This cell line is a good host for studying medical treatments used to address spinal cord and brain trauma. When cultured in vitro, Astrocyte 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 Astrocyte 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. Astrocytes are star-shaped glial cells from the brain and spinal cord that provide support in the blood-barrier to endothelial cell. Specifically, astrocytes aid in biochemical processes such as extracellular K+ level regulation, neurotransmitter removal, and brain/spinal cord repair. Primary astrocyte cells are also known as astroglia. The three types of astrocytes are (1) fibrous, (2) protoplasmic, and (3) radial. Current research indicates that astrocytes release gliotransmitters to communicate with neurons and assist with repair of the brain and spinal cord. As a result, studying astrocytes is vital to the field of neuroscience and in particularly for treatments for spinal cord injuries. Astrocyte 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 Astrocyte cells www.sciencecellonline.com Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

3. Astrocyte Transfection Protocol 1. Plate 10,000 - 15,000 Astrocyte 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 Astrocyte 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 Astrocyte 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 Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

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 Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

5. Astrocyte Transfection Kit Product Details Two component formulation enhances lipid mediated transfection efficiency Optimized easy-to-use transfection protocol provided for transfection of siRNA, DNA, mRNA, and microRNA 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 siRNA 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 Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

6. Data Figure 1. SiRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into astrocyte cells following the recommended protocol. At 48 hours post-transfection the cells were analyzed by qRT-PCR for gene expression levels. 18S rRNA levels were used to normalize the Lamin A/C data. Values are normalized to untreated sample. Data are means ± SD (n=3). Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products

7. Data Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Figure 2. Protein expression of Lamin A/C in Astrocyte cells. DNA plasmid expressing Lamin A/C or siRNA targeting Lamin A/C were transfected into Astrocyte 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.

8. Pre-optimized transfection protocol for Astrocyte 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 Astrocyte 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 Astrocyte cell lines Bio-degradable after endocytosis Used for preclinical research worldwide Astrocyte 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 Products > Astrocyte Transfection Reagent (Primary Astrocyte Cells, Astroglia) Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products