Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) Altogen labs Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) Altogen Biosystems offers the 4T1 Transfection Reagent among a host of 100+ cell line specific In Vitro Transfection Kits. 4T1 Transfection Reagent is an advanced formulation of lipid-based reagents, and it is developed to provide high transfection efficiency with 4T1 and other breast cancer cell lines. This cell line is a good host for studying breast cancer and other cancer types. When cultured in vitro, 4T1 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 4T1 Transfection Kit at www.Altogen.com 4T1 Cell Line www.Wikimedia.org Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

4T1 Cell Line Description and Applications Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) 4T1 Cell Line Description and Applications The 4T1 cell line originates from a mammary gland tumor from a mouse (Mus musculus) that was selected because of its resistance to 6-thioguanine without mutagen treatment. Tumors from this cell line closely mimic the course of human breast cancer, and quickly and easily metastasize and spreads through mice. As a result, this cell line provides an important model in cancer research and for stage IV human breast cancer in particular. Tumors induced by the 4T1 cell line can be used in post-operative and non-surgical models. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com

4T1 Transfection Protocol Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) 4T1 Transfection Protocol 1. Plate 10,000 - 15,000 4T1 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 4T1 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 4T1 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

General Lipoplex-mediated Transfection Mechanism of Action Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) 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

4T1 Transfection Kit Product Details Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) 4T1 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

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) Data Figure 1. Cyclophilin B silencing efficiency was determined by qRT-PCR in the 4T1 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).

Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) Data Figure 2. Protein expression of Cyclophilin B in 4T1 cells. DNA plasmid expressing Cyclophilin B or siRNA targeting Cyclophilin B were transfected into 4T1 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.

4T1 Transfection Kit Benefits Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > 4T1 Transfection Reagent (Breast Cancer Cells, CRL-2539) 4T1 Transfection Kit Benefits Pre-optimized transfection protocol for 4T1 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 4T1 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 4T1 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