I'm facing issues with the tumor dissociation protocol for GBM. I use collagenase/dispase with DNase to dissociate my tumor sample. However, even after incubating it for 60mins, I do not get a complete single-cell solution. I'm worried if I incubate it for a longer time, I might over digest it ( I try to cut the tissue as small as possible before adding the digest solution). Hence, even after filtering with 40uM cell strainer, I see a lot of debris ( I've tried filtering, centrifugation at high speed).
How can I reduce these debris so that my cell solution is clear when I plate them as this is causing all my healthy cells to die. Looking for any useful suggestions for this problem.
Thanks in advance !!
When performing tissue dissociation, it is common to encounter cell debris that can affect the quality of the resulting single-cell suspension. Here are some suggestions to reduce debris and obtain a clear cell solution:
Remember to assess the viability of the cells after each step to ensure that the dissociation process does not compromise cell health. By optimizing the dissociation protocol and implementing these suggestions, you can reduce debris and obtain a clearer cell solution for downstream applications.
To remove cell debris after tissue dissociation, centrifuge the cell suspension at low speed (e.g., 300-500g for 5-10 minutes) to pellet the debris. Carefully transfer the supernatant containing the cells to a new tube, avoiding the debris pellet. Optionally, pass the supernatant through a cell strainer (e.g., 40-70 µm mesh) to further remove any remaining debris.