Hi,
I am trying to transfect 293T cells using 2M CaCl2 and 2X HBS (50 mM HEPES, pH 7.07, 10 mM KCl, 12 mM dextrose, 280 mM NaCl, 1.5 mM Na2PO4) However, some plates, still have particles (probably DNA-CaPO4 particiles) after washing with PBS, 4 hours after transfection and most of cells are round shaped, smaller like dead (or osmotic issue). Some have a clear background and cells look normal. Why cells look like this? Why are there differences among plates? If you help me, I will appreciate.
Thanks
Burak
Thank you for your answers. I solved the problem. Previously, I used to incubate the mixture after mixing plasmid, CaCl2 and HBS for 15-20 min. However, when I skipped incubation (applying the mixture onto cells within 1 min after mixing all), cells are both healthy and transfected now. I think 15-20 min incubation caused bigger DNA-CaPO4 complexes which damaged cells.
Ca Ph is a good old school transfection approach (super cheap), but overall it has a bunch of issues - uncontrollable cell toxicity, a layer of precipitant on top of cells, etc. so I'd advise to switch to lipofectamine 2000 or 3000- for tough cells
Or use branched polyethylenimine, also very cheap and super efficient with 293T cells.
fugene is also a good product. Calcium phosphate or calcium chloride is aggressive and difficult to get good transfection efficiency from in my opinion
I think, 2M CaCl2 is a very high concentration for cell transfection, showing greater cytotoxicity. We use 4mM of CaCl2 for MCF-7 and 4T1 breast cancer cell transfection with limited cytotoxicity and enhanced transfection efficiency. You can try with less concentration of CaCl2 and for image purpose, can wash your cells with 10 mM or higher EDTA for few seconds to chelate the Ca2+. For your reference: Article Knockdown of ROS1 gene sensitizes breast tumor growth to dox...
Thank you for your answers. I solved the problem. Previously, I used to incubate the mixture after mixing plasmid, CaCl2 and HBS for 15-20 min. However, when I skipped incubation (applying the mixture onto cells within 1 min after mixing all), cells are both healthy and transfected now. I think 15-20 min incubation caused bigger DNA-CaPO4 complexes which damaged cells.
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