PC-12 Transfection Reagent: Enhancing Transfection Efficiency with Rat Pheochromocytoma Cells

1. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) Altogen Biosystems offers the PC-12Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. PC-12 Transfection Reagent is a two-component formulation that enhances lipid-mediated transfection efficiency, and it is developed to provide high transfection efficiency with PC-12. This cell line is a good host for studying adrenal glands and related adrenal gland cancers in particular. Other applications include drug discovery, gene expression studies, molecular and cell biology research applications. Purchase PC-12 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 >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) PC-12 Cell Line Description and Applications The PC-12 cell line originates from a transplantable rat adrenal pheochromocytoma and was established in 1976. The PC-12 cell line has a population doubling time is more than two days and will stop dividing and differentiate with a nerve growth factor treatment. In regards to differentiation, PC-12 cells will grow long neuritis and display cellular composition changes. The cells are small, have a rounded morphology, usually form in clusters and have limited cytoplasm. The PC-12 cell line is difficult to transfect, particularly after differentiation. This cell line has applications for studying adrenal glands and cancers related to adrenal glands. High Density PC-12 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 >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) PC-12 Transfection Protocol • Plate 10,000 - 15,000 PC-12 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 PC-12 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 PC-12 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 >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) 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 >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) PC-12 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 >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) Data Figure 1. SiRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into PC-12 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).

7. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) Data Figure 2. Protein expression of Lamin A in PC-12 cells. DNA plasmid expressing Lamin A or siRNA targeting Lamin A were transfected into PC-12 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 >PC-12 Transfection Reagent (Rat Pheochromocytoma Cells) PC-12 Transfection Kit Benefits • Pre-optimized transfection protocol for PC-12 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 PC-12 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 PC-12 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