Pardon me, but it would be great if anyone who had successfully done calcium phosphate transfection on MCF10A cell line can share their protocol/recipes. I would want to compare with mine. I've never transfect MCF10A before. Haven't tried my current protocol yet, I simply asked to study the difference in mechanisms between different protocols. I would want to transfect a plasmid + target gene, with CMV promoter, neomycin resistance and of 12kb total size. Thanks!
Dear Ahmad Hafiz!
Please You look at the article:
Cell culture All human mammaryepithelial cells used were obtained from the American Type Culture Collection (Rockville, MD, USA). MCF10A-VP-EGFP cells were generated previouslybystable transfection of MCF10A cells with the VP-EGFP plasmid in which the human vimentin promoter controls the expression of EGFP (Gilles et al., 1999). MCF-7, T47D, MDA-MB-231, BT549 and Hs578T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS. MCF10A cells were grown in a 1:3 (v/v) mixture of HAM F12 and DMEM, supplemented with 20 mg/ml of adenine, 5 mg/ml of insulin, 0.5 mg/ml of hydrocortisone, 2 ng/ml of EGF, 5 mg/ml of transferrin, 1.5 ng/ml of triiodothyronin and 10% FCS.
For transfection of the siRNA duplexes, 75 000 cells were plated in six-well plates in 2 ml/well of culture medium. At 24 h after plating, Vimentin regulation by SIP1 S Bindels et al 4982 Oncogene the cells were transfected byphosphate calcium precipitation byadding in each well 200 ml of a mixture containing the siRNA duplexes (20 nM), 140 mM NaCl, 0.75 mM Na2HPO4, 6mM glucose, 5 mM KCl, 25 mM HEPES and 125 mM CaCl2. At 24 h after transfection, the cells were extensively washed with PBS and incubated for 48 h in culture medium before theywere harvested for RT–PCR analyses or Western blotting analyses. The transfection of an FITC-labelled control siRNA (Eurogentec, Belgium) was also performed in parallel and revealed an uptake of the siRNA in 100% of the cells
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