Altogen Biosystems  848 Rainbow Blvd #823  L as Vegas  N V  8 9107  U SA

1. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) Altogen Biosystems offers the bEnd-3 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The bEnd-3 Transfection Reagent is an advanced liposome formulation that enhances lipid-mediated transfection, and it has been developed to provide high transfection efficiency with bEnd-3 cells. This cell line is a good host for studying flow of blood. When cultured in vitro, bEnd-3 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 bEnd-3 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

2. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) bEnd-3 Cell Line Description and Applications The bEnd-3 cell line originates from the cerebral cortex/brainof Mus musculus and is a brain endothelial cell line. These cells help to circulate blood and constitute the interior surfaces of blood and lymphatic vessels. The bEnd-3 cells can remove waste from as well as send nutrients to the brain. The bEnd-3 cell line is a useful tool for studying blood flow and impediments to flow such as clots and congestion that can cause disease states such as seizures. High Density bEnd-3 Cells 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

3. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) bEnd-3 Transfection Protocol 1. Plate 10,000 - 15,000 bEnd-3cells 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 bEnd-3 cells (from step 2) 7. Incubate cells at 37ºC in a humidified CO2 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 bEnd-3 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

4. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) 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

5. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) bEnd-3 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

6. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) Data Figure 1. GAPD mRNA levels were quantified using real-time RT-PCR in the bEnd3 cells transfected with siRNAs targeting GAPD or non-silencing siRNA. Forty-eight hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for GAPD mRNA expression levels. Data were normalized against the 18S rRNA signal. Control samples were either mock-transfected or untreated. Values are normalized to untreated sample. Data are means ± SD (n=5).

7. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) Data Figure 2. Protein expression of GAPDH in bEnd-3 cells. DNA plasmid expressing GAPDH or siRNA targeting GAPDH were transfected into bEnd-3 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. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >bEnd-3 Transfection Reagent (Brain Endothelioma) bEnd-3 Transfection Kit Benefits • Pre-optimized transfection protocol for bEnd-3 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 bEnd-3 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 bEnd-3 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