So...
I am finding it difficult to perform flow cytometry analysis of fresh primary cell culture. The cells I'm supposed to be using are FRESH (2 hrs incubation max) primary fibroblasts, extracted from mouse. 2 problems I encountered:
1) when taking fresh cells directly from the cell-extraction procedure (without cell seeding / incubating) - I can't even state a proper gate because the cells' scatter plot is over-spread so it appears there's a lot of debris/non-cell objects left.
2) when taking cells from the cell-extraction procedure > seeding > 2 hrs incubation > wash > trypsin, I can state a reasonable gate of living cells, but the % of fibroblasts among them is rather very low. I can't really tell if fibroblasts' yield is damaged by the short incubation time+wash, or that they are little to begin with (they constitute a small part of the heterogenic primary culture).
Any ideas? has someone tried FACS before with fresh primary cells?
thanks
What tissue are these MSC from?
My group has analyzed primary isolates, but it can be a challenge from tissues with very low frequencies of MSC. You may need to come up with an enrichment method in addition to the plastic adherence. Note, 2 hours may not be enough and maybe negative depletion of the cell population is a better method.
Do you gate fibroblasts purely by size/ scatter or with a fluorescent marker?
If you have space on your panel this may help.
2 hours may not be enough. You may try with different time periods to determine the optimal time for your experiment.
This will be a novel work and very intersting for publication.
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