I am currently working with SiHa cell line. I will have to do PI staining (Flow cytometry), for which I have treated the cells with an alkaloid (Plumbagin) in different doses to check the cell cycle arrest. After the drug treatment for 24 hours, the obtained pellet was in darker shade, whereas the control pellet was in the white colour. and the treated cells are clumping and I am unable to suspend them evenly. I have added ethanol to fix the cells. But still my treated cells are clumping with one another and it is hard to suspend them. what can I do to evenly suspend the cells after fixing them with ethanol.
Thank you
Ethanol denatures proteins, which helps to preserve the cells' structure. If the cells are not properly fixed, they may clump together and be difficult to suspend. Use a gentler agitation method, such as pipetting up and down.
Use a buffer that is compatible with the cells and that will not damage them. Add a small amount of serum to the cell suspension and mix gently. Repeat as necessary.
Hello M.Tamil Mani Subi
If the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Therefore, sample preparation becomes the critical first step in any flow cytometry experiment.
I have mentioned a few suggestions below which you could follow on how to avoid cell clumping.
1. The dead cells release DNA in the medium. DNA being sticky in nature can form cell aggregates by causing cells and debris to stick to one another forming clumps. If cells appear clumpy, calculate the volume of DNase I solution that should be added to the sample to yield a final concentration of 100 μg/mL DNase I. Add DNase I solution dropwise to the cell suspension while gently swirling the tube. Incubate at room temperature for 15 minutes. Wash the cells with culture medium or buffer containing 2% FBS. Gently invert to mix, then centrifuge at 300 x g for 10 minutes at room temperature. Discard as much of the supernatant as possible, then gently resuspend the pellet. If cells still appear clumpy, then you will have to pass the sample through a 37 - 70 µm cell strainer into a fresh conical tube. Rinse the sample tube three times with the culture medium or buffer containing 2% FBS, then pass through the strainer.
2. You need to know that calcium and magnesum can promote cell clumping. Use Ca++ and Mg++ free PBS for your staining buffer, and consider adding 1mM EDTA to your buffers as well.
3. If you pellet cells too hard, they can clump. Ensure that cells are not subjected to high centrifugal force.
Hope these above suggestions may help you get rid of cell clumps.
Best.
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