Hi everyone,
I want to fix osteoarthritic chondrocytes in 6-well plate with paraformaldehyde.
Before fixation, it takes at least three days for the chondrocytes to adhere to the surface of the well, but in monolayer culture chondrocytes dedifferentiate in fibroblast-like cells.
I have to preserve my cellular morphology natural (not dedifferentiated form) so, can anyone suggest a protocol that keep the morphology immediately after chondrocyte isolation?
If I am trying to understand your question
You want to fix freshly isolated chondrocytes?
Dedifferentiation is a characteristic trait we notice with chondrocytes not only with adherent cultures, but also non-adherent/suspension cultures as early as 24 hours.
One option would be isolate the cells, spin them down , give a pbs wash and fix with 4% paraformaldehyde for 15 min and wash again
However , this would depend on what is your evaluation parameter?
Thank you Elizabeth Vinod. As you mentioned, I want to fix freshly isolated chondrocytes before they dedifferentiate.
So if I understand correctly, I can fix the cell sediment even if they are not attached to the surface.
In the following, I want to prepare a pattern from the morphology of cells.
@hanie motahar
Yes you can
Once isolated
Centrifuge the cells in an eppendorf or centrifuge tube ... Would depend on your cell...say 1200 rpm for 5 min
Next remove supernatant
Give a wash with 1x PBS
Again supernatant...
Add 1 ml of 4% paraformaldehyde ...gentle titurate ... Leave 15-20 min
Add PBS...centrifuge ...to wash....reconstitute with new PBS... And analyze
Elizabeth Vinod
Thank you so much for your patient response. I will try.
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