1. Products > U87 Transfection Reagent (Glioblastoma Cells) Altogen Biosystems offers the U87 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The U87 Transfection Reagent is an advanced formulation of a lipid based reagent, and it has been developed to provide high transfection efficiency with the U87 cell line. This cell line is used for researching cytotoxicity of chemotherapies as well as for researching gliobastoma. When cultured in vitro, U87 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 U87 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. U87 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 High Density U87 cells Atcc.org Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > U87 Transfection Reagent (Glioblastoma Cells) The U87 cell line is crucial to investigating the toxicity of chemotherapies aimed at cancer as well as for investigating the in vitro model of gliobastoma cells. Stage 4 gliobastoma or gliobastoma multiform (GBM) is a deadly brain cancer; it is a primary malignant brain tumor and there are approximately 16,000 new diagnoses yearly in the United States alone. Furthermore, GBM only has a 29.6 % one-year survival rate. The U87 cell line was established from a 44-year old Caucasian female with Stage 3 GBM. The cell line has epithelial morphology and has a modal chromosome number of 44. All of the U87 cells have 12 makers in common and N1, N6 and N9 were not found.

3. U87 Transfection Protocol 1. Plate 10,000 - 15,000 U87 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 U87 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 U87 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 > U87 Transfection Reagent (Glioblastoma 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 > U87 Transfection Reagent (Glioblastoma Cells)

5. U87 Transfection Kit Product Details Two component formulation enhances lipid mediated transfection efficiency Optimized for intracellular delivery of plasmid DNA, siRNA, microRNA, and mRNA High transfection efficiency of both siRNA and plasmid DNA without compromising cell viability Achieve robust siRNA uptake for dependable gene silencing Effective transfection under conditions of up to 40% serum Transfection kit includes Transfection Enhancer reagent Gentle enough to be used for single cell analysis 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 > U87 Transfection Reagent (Glioblastoma Cells)

6. Data Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Figure 1. GAPD mRNA levels were quantified using real-time RT-PCR in the U87 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). Products > U87 Transfection Reagent (Glioblastoma 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 GAPDH in U87 cells. DNA plasmid expressing GAPDH or siRNA targeting GAPDH were transfected into U87 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 > U87 Transfection Reagent (Glioblastoma Cells)

8. Pre-optimized transfection protocol for U87 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 U87 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 U87 cell lines Bio-degradable after endocytosis Used for preclinical research worldwide U87 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 > U87 Transfection Reagent (Glioblastoma Cells)