1. Products > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427) Altogen Biosystems offers the MG-63 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The MG-63 Transfection Reagent is an advanced formulation of a lipid based reagent, and it has been developed to provide high transfection efficiency with the MG-63 cell line. This cell line is an excellent transfection host and can be useful in a wide array of biomedical applications. When cultured in vitro, MG-63 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 MG-63 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. MG-63 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 MG-63 cells www.lonza.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427) The MG-63 cell line originates from the bone tissue from a 14-year-old Caucasian male with osteosarcoma. The cell line is hypotriploid and has a modal chromosome number of 66. The MG-63 cell line displays fibroblast morphology and express TGF-beta RI and RII (transforming growth factor beta). The cell line produces high levels of interferon with stimulation. The interferon is induced via cycloheximide, actinomycin D and polyinosinic-polycytidylic acid. The MG-63 cell line is an effective transfection host and has applications in a wide array in the biomedical field.

3. MG-63 Transfection Protocol 1. Plate 10,000 - 15,000 MG-63 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 MG-63 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 MG-63 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 > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427)

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 > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427)

5. MG-63 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 > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427)

6. Data Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Figure 1. siRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into MG63 cells following the recommended protocol. At 48 hours post-transfection the cells were analyzed by qRT-PCR for Lamin A/C 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=4). Products > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427)

7. Data Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Figure 2. Protein expression of Lamin A in MG-63 cells. DNA plasmid expressing Lamin A or siRNA targeting Lamin A were transfected into MG-63 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 > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427)

8. Pre-optimized transfection protocol for MG-63 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 MG-63 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 MG-63 cell lines Bio-degradable after endocytosis Used for preclinical research worldwide MG-63 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 > MG-63 Transfection Reagent (Osteosarcoma, CRL-1427)