Dear all,
I am straggling with Ca transfection on neurons. I heared that antibiotics like Penicillin will reduce the transfection efficiency. My transfection medium do not contain antibiotics, but the plating medium which neurons live have 1% Penicillin. Does that also matter? I am suffering from the low transfection efficiency. Any suggestion is appreciated!
Thanks all.
Hi, Here is a protocol I developped during my master:
For more detail, see on pubmed: Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis
About Ca transfection, the pH is very important and the good pH may vary depending the lab, the day... So when you prepare HBS, you should prepare several pH (6.8, 7, 7.2,7.4 for example), test them and keep the best. The precipitate should louk like a very little...
CGN grown on glass coverslips in 24-well plates were transfected at DIV 6 with pCI expression vectors encoding GFP (as a control) or different forms of Trim17 fused to GFP. For this purpose, we used a calcium phosphate protocol that has been optimized for neuronal cultures by the group of Dr M.E. Greenberg,39 with slight modifications. Briefly, the conditioned culture medium was removed, filtered and saved. The neurons were washed twice and incubated for 1 h with DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 25 mM KCl, 1 μM (+)-MK-801, 10 mM MgCl2 and 5 mM HEPES, pH = 7.5. During this time, the DNA/calcium phosphate precipitate was prepared by mixing one volume of DNA in 250 mM CaCl2 with an equal volume of 2 × HBS (274 mM NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 15 mM d-glucose, 42 mM HEPES, pH 7.0). Two μg of plasmid DNA in 30 μl of CaCl2, mixed with 30 μl 2 × HBS, was used per well. The precipitate was allowed to form for 30 min at room temperature. Then, 60 μl of the DNA/calcium phosphate precipitate were added drop-wise to each well and cultures were returned to the CO2 incubator for 50 to 60 min. The incubation was stopped by “shocking” neurons for 1 min with 1 × HBS supplemented with 10 mM MgCl2, 2% DMSO and 5 mM HEPES, pH = 7.5. CGN were then washed twice with DMEM supplemented with 25 mM KCl and 1 μM (+)-MK-801. The saved conditioned medium was added back to each well and the cultures were returned to the 5% CO2 incubator at 37°C. This protocol allows a transfection efficiency of up to 15% with minimal toxicity. Sixteen h after transfection, CGN were incubated in K5 medium for 8 h or left untreated. CGN were fixed, stained with Hoechst 33258 and analyzed by immunofluorescence in some experiments. Coverslips were mounted on glass slides in Mowiol and transfected neurons were detected by GFP fluorescence. Among GFP-positive neurons, apoptosis was assessed by observing the morphology of the cell and detection of nuclear condensation or fragmentation by Hoechst staining. For each experiment and each condition, at least 200 GFP-positive neurons were scored in a blinded manner. Statistical analyses were performed using GraphPad InStat version 3.0 for Mac (GraphPad Software, San Diego California USA, www.graphpad.com).
Hello Dear Chenglong Yu,
The streight forward answer to your question is, Yes (IT SHOULD BE ALL ANTIBIOTIC FREE), antibiotics do affect the transfection efficiency so you need to use the antibiotic free media. During normal cultivation of your neurons of interest, you can grow the cells with normal antibiotic supplementation. But after you seed the cells into plates, you have to grow them in antibiotic free media only. Likewise you can grow your plated cells in antibiotic free media for 24 hrs and then you can proceed for your transfection experiment (all experiment should be in antibiotic free media). You can easily opt for the same because cells don't catch contamination so soon even in the antibiotic free media if handled properly..
Hope it helps !
Dear Ajay,
Thank you for the clear answer. I guess I need to grow neurons in antibiotic free medium. Previously I succeed in Ca transfection when I do so, but now I see many neurons die after transfection when I add penicillin in the cultivation these days.
Thanks a lot. @Ajay Kumar
Dear Laurent Vanhille,
Thank you for sharing. I wonder why you need MK-801 and MgCl2 during incubation? Does NMDAR affect the transfection efficiency?
Another question is that, after adding DNA/calcium phosphate precipitate, do you change the medium or just use the same one with MK-801 and MgCl2?
Thanks a lot. @Laurent Vanhille
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