I never used hepes for buffering ph and never had this problem with these cells for years. Pics are for A549 in wells (looking good before changing media) and looking rounded and bubbley once media is removed, washed with pbs and added new media. I do all steps extra carefully and slowly and still problem persists-and previoously i never had to work slow they were fine. media is DMEM, 1% pen strep 10% FBS, 6mM glutamine (we have been using these concentrations for long, with no prob)
I tried different cell stocks (from liquid nitrogen stocks prepared by different people, and different passage numbers), new reagents for everything in the complete media and PBS, cells grow fine in flasks, but in dishes once media is changed cells act like this. What do you advise?
Maybe the problem is with dish? Maybe you have a bad batch (dishes are not evenly coated), so are no suitable for adherent cells?
Is the pH of your media right? When I cultured cells (many years ago now) we used a pH indicator in the media, and we would bubble (sterile filtered) CO2 into our media periodically to adjust the pH. The media was pH buffered with Calcium carbonate and our cell incubators used some added CO2 in the atmosphere. Media stored in the refrigerator would over time become alkaline as it lost CO2 (which forms carbonic acid in water) and so we bubbled in the CO2 to bring it back to the right pH before using it on cells. I never applied alkaline media and observed the reaction of the cells, but it seems this could be a possible reaction to a pH change.
1. Please double check the conditions of incubator,
2. Do not use any cold reagent
3. I have also observed this difference in A549, and I found that if we use cold PBS then it happens like this.
4. in our lab we usually use 1640 for A549
Thank you angieszka but I tried using dishes from lab mates who don't have a problem and I had the same issue.
Thank you Brian I used media from lab mates who have no problem and problem persisted.
Thank you Ghulam I warm everything before working and take then out of water bath, disinfect and directly work so they're still warm.
I, feel now it's something with the cells since everything else when borrowed still problem persists. But I already tried several cell stocks prepared by different people.. I don't know what else to try
If your cells are growing fine, in flasks, you must look at the dishes and/or your process. In earnest, I suspect your process. Take a completely unrelated set of cells - try a solid line of epithelial or other well-known stock, and apply your same process, media, dishes, et al. Run through a few cycles of this and note your results. There are no magical mysteries in cell culturing. Your materials or your process are awry, unless you find that an ordinary culture of epithelial cells, from another lab, is normal. Then, you have to begin looking at your cell lines, and potential for infected colonies.
One other thing: when was the last time you scrubbed down your cell culture room, and everything in it? The Scientific Method will never fail you. Keep hammering away at the variables, and you will identify your root cause.
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