I am trying to lift, dissociate, and then flow fixed HeLa cells for fluorescence analysis. When doing this previously, the cells lift from the plate with 0.5% trypsin-EDTA treatment but they then remain attached to each other and clump up in the flow cytometry buffer upon resuspension. Is there a treatment to dissociate the cells from each other, or an overall better process to attain a single cell suspension?
You can wash with PBS by mild centrifuging after trypsinization and don't keep cells with trypsin for long. Carefully pipette cells up and down before doing flow cytometry. You can also try with small amount of cells because full confluency sometimes often creates this problem.
You should dissociate the cells with cold PBS or accutase (which is on-ionic reagent), which does not cause any harm to cell membrane like trypsin-EDTA.
Hello,
I used two 2 trypsin treatments, first one to inactive the FBS for less than a minute and 2nd one for few minutes to detach the cells. Before labelling, 2-3X PBS wash with gentle pipetting. In my experience, using 2X Trypsin wash with gentle tapping of the cell plate/flask to detach cells allowed for high degree (>90%) cell viability as assessed via 7-AAD and annexin staining. This protocol worked even for cells with precoated cultures such as those of cultured cardiomyocytes in my experiences.
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