The cells are suspended in PBS with the IP and RNase for the study of the cell cycle but they form aggregates. I tried to dilute and filter but the cells still form aggregates
Hey Marie,
De maniere generale on utlise entre 5 et 10 mM EDTA dans du PBS pour decoller des cellules d'une boite sans utiliser de trypsine donc je commencerais par quelque chose du genre. Assure toi, mais je suis sur que tu y a deja pense, qu'il n'y a pas de Calcium dans ton PBS et rince bien tes cellules 3-4 fois pour enlever un maximum du calcium du milieu... Dis nous ce que ca donne. Cheers
Dear Marilyne Guillamin,
Usually,aggregation occurs in the fixtion step(ethanol adding) and that s might affect results accuracy especially when carrying a FACS analysis. To avoid aggregation, try to separate cells by tabbing or by a gentle pipetting. If not you might use a 25G syringe to separate cells mechanically. In this case, it would be harmful to cells if not applied approprietely.
I hope it can help.
Hamadi.
Not a cell type I am familiar with, but in general I would try two options:
1. This may be due to cell lysis. If possible reduce the time between seeding and harvesting. The longer in culture, the more ECM some cells produce and therefore harvesting needs to be harsher, leading to lysis and aggregation. This could also be helped by adding DNAse rather than RNase
2. Add some protein like BSA.
finally i don't use EDTA but more action with pipet and if necessary i use needlee to break cells aggregates
Hi Marilyne,
We use 1-5 mM of EDTA to avoid clumps. We equaliy saw that the use of DPBS for cell preparation to FACS analysis induce aggregate. Soon as possible, we centrifuge cells with FACS buffer (with FBS or BSA). If the suspension continue to aggregate, it could be due to a DNA release during apoptosis and a 10 min DNAse treatment could solve the problem. Nevertheless, the DNAse function need the présence of cations (Ca+/Mg+).
Good luck for your experimentations Marilyne.
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