Hi everyone,
We are trying to optimize a protocole for cell cycle analysis in 6-well plates. We prefer this format of plates because it is what we use regularly for siRNA transfections. So we wouldn't like to change the size of plates.
We have a protocol using PI in which we fix the cells with ethanol. We lose almost 90% of cells after this fixation.
Could you give me tips for avoiding this loss of cells?
Thanks.
Dear Dr.Chaveroux,
We prefer to trypsinize and harvest cells, before 70% EtOH fixation. The protocol in our institution is not based on the direct operation on 6-well plates.
Good luck and I hope this helped.
Dear Dr.Chaveroux, do you want to keep cells on the plates? What excat protocol do you use?
Ethanol rapidly changes surface properties, so, cell easy de.attach from the plates. As alternative, you can try formaldehyde but this dependent from your protocol.
My best wishes!
Dear Dr.Chaveroux,
I never heard fixation on the plate for cell cycle analysis purpose.
If possible in your experiment, I suggest you to harvest cells, wash them twice, fix in cold 70% EtOH and keep at +4° almost 3-4 hours before PI staining. If you work in 1.5ml tubes, you can wash cells by suction to avoid lost of cells.
Good luck!
feedback of the last experiment. We indeed improved the amount of cells used for FACS. One tip is to centrifuge with swinging buckets instead of a fixed angle centrifuge after EtOH fixation. However the loss of cells remains important. Any other protocol or advice for augmenting the number of cells following fixation?
Thanks
Hello!!
May be I can help, We use the 6-well format. But we don't use FACS, instead we have the Muse cells analyser, I don't know if the final volume of cells with PI is the same. We seed 300.000 cell/well for a 24 hour experiment and every well is used for one lecture, so for each experiment we use 3 wells to have the minimum of repetitions.
Protocol
1.- Harvest your cells with your favorite tripsin format, we use the 0.05%. For my mammary cells (MCF7/MDA-MB-231) its took about 5 min.
2.- Centrifuge your cells for 10 min at 300 g in 15 ml tube.
3.- Remove the supernatant, add 1ml of pbs and put it in a 1.5 ml tube. Repite the centrifuge.
4.- Remove the PBS with a pipete and repite the process. When my pellet its not good enough and I can see rest of cell in the wall, I do a quick spin at max revolution.
5.- Remove the PBS, but leave 50 ul of PBS, resuspend your cells and add drop by drop in 1mL of 70% Ice-cold EtOH. Let in -20ºC for at least 3 hours. We left it overnight.
6.- In this step we always lost a lot of cells. We took all the fixed cells in EtOH and centrifuge in a 15 mL tube, its is important to use this type of tube for avoiding cell lost . Centrifuge for 5 min at 300 g.
7.- Wash them in 250 uL of PBS (in the same tube) for 10 min at 300g.
8.- Carefully remove the pbs and add 250 ul of your PI solution. wait 30 min at dark and read. We read about 10.000 events.
The protocol of cell cycle kit of Muse, encourage us to use a 15 ml tube after the fix process because in your wash step you always lose a huge amount of cells if you use a lower volume tube.
My apologies for the grammatical mistakes, I do my best. I hope these tips can help you.
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