ARPE-19 Transfection Reagent for Efficient Retinal Epithelium Transfection

1. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >ARPE-19 Transfection Reagent (Retinal Epithelium) Altogen Biosystems offers the ARPE-19 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. ARPE-19 Transfection Reagent is an advanced cationic lipid-based formulation, and it is developed to provide high transfection efficiency with ARPE-19 and other cell lines. This cell line is an excellent host for transfection. When cultured in vitro, ARPE-19 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 ARPE-19 Transfection Kit at www.Altogen.com ARPE-19 Cell Line Sciencecellonline.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 >ARPE-19 Transfection Reagent (Retinal Epithelium) ARPE-19 Cell Line Description and Applications The ARPE-19 cell line originates from the retinal pigment epithelia (RPE) of the healthy eyes of a 19-year old male. The cells were derived by Amy Aotaki-Keen in 1986 after the man died from a head trauma from a motor vehicle accident. After subjecting the cells to selective trypsinization for four passages in order to remove superficial cells, the cells were run through a fifth passage and displayed rapidly growing retinal pigment epithelia. These fast-growing RPE cells formed cobblestone monolayers and also became pigmented after several months. ARPE-19 cells are particularly useful as a tool for transfection. In fact, due to RPE-specific markers (CRALBP and RPE-65), this cell line is especially valuable for cellular and molecular biology research. 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 >ARPE-19 Transfection Reagent (Retinal Epithelium) ARPE-19 Transfection Protocol 1. Plate 10,000 - 15,000 ARPE-19 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 ARPE-19 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 ARPE-19 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 >ARPE-19 Transfection Reagent (Retinal Epithelium) 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 >ARPE-19 Transfection Reagent (Retinal Epithelium) ARPE-19 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 >ARPE-19 Transfection Reagent (Retinal Epithelium) Data Figure 1. siRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into ARPE-19 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).

7. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products >ARPE-19 Transfection Reagent (Retinal Epithelium) Data Figure 2. Protein expression of Lamin A/C in ARPE-19 cells. DNA plasmid expressing Lamin A/C or siRNA targeting Lamin A/C were transfected into ARPE-19 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 >ARPE-19 Transfection Reagent (Retinal Epithelium) ARPE-19 Transfection Kit Benefits • Pre-optimized transfection protocol for ARPE-19 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 ARPE-19 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 ARPE-19 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