Is it even possible to restore and reuse buffy coat for PCRs and also for flow cytometry?
An easy and convenient way to go about would be -
a] for PCR: you can isolate the DNA and store it accordingly. DNA is a stable molecule and you can use it whenever you want.
b]for cytometry: you can fix the cells and store them easily. You can go through the normal steps of preparing cells for cytometry and just stop before the staining step, Suspend them in the wash buffer you use (the step of washing after permeabilization ) and store them at -80 until further use, which hopefully would not be after a long gap.
Appreciated. But is it gonna be a direct way of preserving and storing buffy coat it self ?
In my opinion you can just fix the cells and keep them in your -80. You just want to store them and not use them for other live cell experiments, so in that case that should suffice.
Storing the cells in -80 would damage the cells due to ice crystal formation and also its not advisable to fix the cells before staining for the flow cytometry protocol. always staining first and fixing later is recommended unless you have a specific reason to do vice versa.
Heres what you can try Mr. Harindra:
For PCR:
If you are talking about reverse transcriptase PCR, you can store the samples at the following steps of RNA isolation:
a: add 1 ml of TRI reagent to your PBMC suspension and aspirate till its a clear soultion and leave it in 4 degrees for an hour and then transfer it to -80 degrees until further use. (this way, the RNA can be stable for a month)
b: you can also stop the protocol at isopropanol step or ethanol step and store the sample.
c: you can go all the way till c-DNA and store the c-DNA. Its not preferable to stop at the RNA step and store the RNA. It will degrade easily.
If you are talking about a normal PCR from the genomic DNA, what Raunaq suggested will do. Just isolate the genomic DNA and store it at - 80 until further use.
For flow cytometry:
After you isolate the PBMCs from the buffycoat, just finish the staining protocol and finally fix the stained cells with 300 microlitres of 2% paraformaldehyde in PBS. (or whatever fixing solution you are using specifically) at room temperature for 10 minutes.
One important thing you have to remember here is, if you are using paraformaldehyde (PFA), make sure that the solution is at 37 degrees when you add it to the cells. Keep the 2% PFA solution in the water bath at 37 degrees for some time before using it. This is because, The PFA at lower temperatures will crumble the cells and the forward scatter and side scatter will not be perfect.
One more thing is please DO NOT acquire the sample while it is PFA, because PFA will show some auto fluorescence in FITC channel if I am not wrong.
After the fixing step in PFA, spin down the cells again and re suspend them in 1X PBS (usually 300 microlitres). and leave them in 4 degrees until acquisition.
However even after fixing, you have to acquire the data within one week because the fluorescence starts quenching after that.
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