My cell cultures (HeLa S3, mouse kidney cells, WB-neo, WB-ras) strarted to form clumps during passaging so it is not possible to count them properly.
I TRIED
1. make PBS without Mg and Ca ions,
2. add EDTA to trypsin
3. use more diluted trypsin
4. reduce exposure time to trypsin to 3 minutes
Nothing has helped in clumps formation.
Would anyone know what could be the reason and how to solve it?
Thank you very much. Kateřina Delawská
Since it is more than one cell type, that suggests a method problem rather than a change in cells. Do the opposite for # 3 and/or #4. Try using LESS diluted trypsin. That is, a more concentrated trypsin. Alternatively, or in addition, try a longer trypsin exposure time.
Hi,
Some cell types are building massive amounts of Extracellular matrix. For me it always worked best to Add Trypsin EDTA SOlution and leave them in the incubator for 10-15 or even 20 minutes untill I saw that the cells dislodge. Do not shake prior to reaching this state
Good luck
Sven
Dear Katerina, use quite cold PBS (Ca-Mg free) to wash your cells. Try to replace your cell culture plasticware (flasks, plates). Since, it is happening with all of your cell lines, so may be problem is within the material of flasks or plates. Since it is well known that over trypsinization causes extreme clumping and you are not doing it so my doubts are towards your plasticware. You can also perform vigorous pipetting other than normal during cells splitting to get rid of heavy clumps.
Also see whether your Trypsin is properly made and is not very concentrated due to some wrong calculations. If you can purchase Versene-EDTA (a gentle non-enzymatic, but very effective cell dissociator) to replace Trypsin and just see what will happen to clumping.
Also if your plastic ware is not the culprit you can try to use 2.5% Trypsin-EDTA during passage rather than 0.5% or 0.25% Trypsin.
TrypLE (Gibco) is another Trypsin alternate, which is animal origin free, maybe it will work fine with your cells, as it is less damaging than Trypsin.
Sincerely....
Ron Hrstka I tried to use less concentrated trypsin and shorter exposure time this time because I was normally using more concentrated trypsin for longer exposure time. But anyway thank you for your suggestion.
Sven Reister Dear Sven, it is generally known that it is not good for cells to be in trypsin for longer time than 10 minutes..
Jawwad Ahmad We are currently using different plastic ware than we were before, so it could be that problem. Thank you for your answer!