MDCK Transfection Reagent for Efficient Transfection of Kidney Cells

1. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > MDCK Transfection Reagent (Kidney Cells, CCL-34) Altogen Biosystems offers the MDCK Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. The MDCK Transfection Reagent is an advanced formulation of a biodegradable polymer-basedreagent, and it has been developed to provide high transfection efficiency with the MDCK cell line. This cell line is a good host for studying kidney cell function. When cultured in vitro, MDCK 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 MDCK 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 > MDCK Transfection Reagent (Kidney Cells, CCL-34) MDCK Cell Line Description and Applications The MDCK cell line was established from kidney cells of a healthy cocker spaniel dog (Canis familiaris) in 1958. The MDCK cell line exhibits epithelial morphology and tests positively for keratin. Regarding research purposes, the MDCK cell line makes an excellent transfection host, but is susceptible to viral infection. Generally, this cell line has application for research relating to kidney cell functioning as well as biomedical gene expression. The cell line also has applications for the processing of the beta-amyloid precursor protein and subsequent proteins. High Density MDCK cells www.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 > MDCK Transfection Reagent (Kidney Cells, CCL-34) MDCK Transfection Protocol 1. Plate 10,000 - 15,000 MDCK 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 MDCK 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 MDCK 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 > MDCK Transfection Reagent (Kidney Cells, CCL-34) 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 > MDCK Transfection Reagent (Kidney Cells, CCL-34)- MDCK 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 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

6. Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > MDCK Transfection Reagent (Kidney Cells, CCL-34) Data Figure 1. siRNAs targeting Lamin A/C mRNA or non-silencing control siRNA were transfected into MDCK 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 > MDCK Transfection Reagent (Kidney Cells, CCL-34) Data Figure 2. Protein expression of Lamin A/C in MDCK cells. DNA plasmid expressing Lamin A/C or siRNA targeting Lamin A/C were transfected into MDCK 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 > MDCK Transfection Reagent (Kidney Cells, CCL-34) MDCK Transfection Kit Benefits • Pre-optimized transfection protocol for MDCK 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 MDCK 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 MDCK 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