I am growing in L15 media at 37 deg C without CO2 as recommended by ATCC and carrying out transfection using lipofectamine 2000 with PLUS. But i observe rounding up of cells and very poor transfection efficiency.
we generally use DMEM medium with 10% FBS.. and with 5% CO2. Please try to place the cells in CO2 incubator..
We have grown MDAMB231 both in L15 without CO2 and in DMEM with 5% CO2. We found that the cells look healthier and grow faster in DMEM. This is likely due to the fact that DMEM is very rich (e.g. high glucose concentration).
I would think that it really doesn't matter much what media you use, as long as your experiments are kept consistent (i.e. you use the same media and growth conditions for all experiments).
Thanks Mario, could you also please suggest a workable transfection protocol as we are struggling to get them transfected. As i have mentioned we use Lipofectamine 2000 with PLUS.
I am sorry Merlyn. There is no easy answer.
Experience has shown that the only reasonable way is to systematically test the relevant variables in your system empirically. That means testing different amounts of DNA as well as different DNA to LF2000 ratios. I have also seen significant differences in transfection efficiency depending on cell density. Once you set up a systematic matrix and test it with a reporter (like GFP) then you can pick the best condition (based on survival or transfection efficiency) for your actual experiment.
We are also using DMEM (high glucose), 10% FBS. Previously we used L-15, but we switched to DMEM, as the morphology and growth was better, just as Mario described.
I wanted to add that as L-15 is optimized for air without added CO2. The buffer system in L-15 is made for air with ambient CO2. If L-15 is put @ 5% CO2, the medium gets quite acidic, as you can imagine this is not especially good for the cells. So I would not recommend putting cells with L-15 in a 5% CO2 incubator.
thank you all for your suggestions.
We actively do imaging on our cells and we were in particular facing problems related to their morphology after transfection. I believe that not very high transfection efficiency can be achieved for MDA MB 231. we did try growing them in DMEM but we were seeing too many large acidic vacuoles. is it particularly due to the media? I m interested to know whether is it a very specific problem that we are facing or is it a very general problem.
DMEM with FBS is the ideal choice with 5% Co2, However hypoxic conditions induce variable characteristics. So There is no direct answer, you need to be specific of what output you want and then tailor the medium to get desired results.
Beware, all DMEM is not created equal. ATCC has formulated their DMEM for use with 5% CO2 by adding only 1500mg/L of NaHCO3 while most others are formulated with 3700mg/L, optimized for 10% CO2. We purchased DMEM without NaHCO3 and added it ourselves.
I have grown MDA MB 231 cells in DMEM with no trouble but did not try transfecting them.
I agree with Audrey, you have to be careful which DMEM you choose as there are a number of different variants with different glucose, pyruvate and NaHCO3 concentrations. If you do not want to use CO2, try DMEM with HEPES. What happens to your cells with L15 versus DMEM. Also what happens with your lipofectamine alone control (i.e. no plasmid)? Transfection does affect cell viability, I use Effectene from Qiagen, the efficiency is good, but the effect on the cell viability is much lower.
Dear Andrew
well my cell in L15 grow and attach well as compared to DMEM but the transfection efficiency is very low and cells which are transfected get round up and loose their morphology. In my mock treated cells i still do see cells rounding up and floating. i have been using Lipo2000 with PLUS. I m thinking to try Lipo 3000 from invitrogen.
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