Hi,
I'm having some troubles when seeding cells in m24 plates: after 24-48 hours of seeding, cells don't seem to attach in some areas of the wells or they attach at the beginning but then they die. This leads to irregular replicates and no-reliable results. I've observed this in 2 different cell lines (sw620 and DLD1), I've changed medium bottles, tried with different cell vials, and put plates in 2 different incubators, but I still have this irregular attachment/growing. Could you help me? I've done lots of this experiments before, both in m24 and m96, and everything went OK. I don't know what can be failing now...
Thank you!
maybe the plastic of these multiwellplate is not so good or the "tissue treatment" were bad. It can be a process error by manufacturer. try with other 24plates...
Explain to me exactly how you are seeding the well step by step. I might have a few ideas I can share.
Please check your seeding density; too many cells in a well might lead to death in some cell lines. Also, check the quality of the plate; some plates may not be suitable for seeding cells and might require growing the cells on treated cover slips in the wells.
The causes can be multiple, colleagues cited the quality of the plates, see also the conservation mode, their aging.
The cell distribution operation is quite long exposing your cells to heatand deposition of cells in the vial containing the initial suspension ( unequal distribution.
Test the viability of cells in your working condition concomittente way): a portion of the cells will be placed in T25 flasks will be incubated in the presence and absence of CO2.
The causes can be multiple,
Hi, I suggest you to use non-pyrogenic polystyrene coated tissue culture graded well plates. Please refer http://www.nestscientificusa.com/products_detail/&productId=26.html. Also kindly check out your seeding density or confluency % during seeding. Kindly increase the serum concentration if the problem exists again
Are you by any chance using plates that were sterilized with ethylene oxide? Residual ethylene oxide can kill cells. Look for plates that were radiation-sterilized or - better - manufactured and packaged under sterile conditions.
Hi everyone,
first of all thanks for all answers. I have used these seeding protocol and cell density other times, so I know they work. I think cell viability is ok since they attach and grow well in p60 and p100 dishes....
We have already used these plates before, from the same supplier...
Since your cells grow well in 60-mm and 100-mm dishes (presumably using the same culture medium), I would try tissue culture 24-well plates from different manufacturers. We once compared culture dishes from different manufacturer and found huge differences in adhesive properties although the dishes were advertised as identical.
Thanks for the new answers! I'm using DMEM with 10% FBS. The thing is that I've used same plates and medium before, and everything went OK.
Troubles started when we moved to other building. First we thought that it could be something in the CO2 of the incubators, since conductions were new and they could have some dirt, but we connected to a gas bottle and nothing changed; then we suspected the vapours of the bleach we were using to clean, but we stopped using it (we now se Virkon) and troubles continued. The other thing that has changed since we've moved is that now we have to keep the vaccuum system used to aspirate medium inside the flow cabinet, including the bottle with the aspìrated medium and virkon. So we are now wondering if some vapour could be escaping from the bottle and affecting cells....
Hi Lauriane,
All the incubators and some of the hoods are the same we used in the other building, we moved them here....
Thanks!
Thanks Laurianne! we were thinking about any gas could be produced in the bottle but we haven't realized it could be also generating vibrations.
We are going to change the vaccuum system so it can be placed outside, so I'll tell you how it turns out.
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