L6 Transfection Reagent for Rat Myoblast Cells

1. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) Altogen Biosystems offers the L6Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. L6 Transfection Reagent is an advanced formulation of a biodegradable polymer-based transfection agent, and it is developed to provide high transfection efficiency with L6 and other mammalian fibroblast cell lines. This cell line is a good host for studying mammalian tissue and connective tissue in particular. Other applications include drug discovery, gene expression studies, molecular and cell biology research applications. Purchase L6 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 >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) L6 Cell Line Description and Applications The L6 cell line originates from the fibroblasts of rat (Rattus norvegicus) thigh muscles, and it is a particularly effective tool for studying connective and mammalian tissue. This cell line was isolated by Yaffe and passaged in methylcholanthrene. The L6 cell line displays myoblast morphology and appears similar to skeletal muscle tissue. The cell line has several unique properties including the ability to fuse, the ability to create multinucleated myotubes and striated fibers as well as a propensity for high cellular variability. Due to the unique properties of L6, the cell line is frequently utilized in biological research studies. High Density L6 Cell Line 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 >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) L6 Transfection Protocol • Plate 10,000 - 15,000 L6 cells per well in 0.5 ml of complete growth medium 12–24 hours prior to transfection • Wash with 1xPBS and add 0.5 ml of fresh growth medium • 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 • Incubate transfection complexes at RT for 15 - 30 minutes • 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 • Add prepared transfection complexes to 0.5 ml of complete growth medium with L6 cells (from step 2) • Incubate cells at 37ºC in a humidified CO2 incubator • 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 L6 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 >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) 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 >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) L6 Transfection Kit Product Details • A biodegradable polymer-based transfection agent – once inside the cell, the polymer degrades into smaller less toxic components reducing cell toxicity, facilitating release of the transgene, and improving 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 >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) Data Figure 1. Cyclophilin B silencing efficiency was determined by qRT-PCR in the L6 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=6).

7. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) Data Figure 2. Protein expression of Cyclophilin B in L6 cells. DNA plasmid expressing Cyclophilin B or siRNA targeting Cyclophilin B were transfected into L6 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 >L6 Transfection Reagent (Rat Myoblast Cells, CRL-1458) L6 Transfection Kit Benefits • Pre-optimized transfection protocol for L6 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 L6 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 L6 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