I want to measure cell cycle after a chemotherapy treatment of a B lymphocyte cell line. It may cause cell death.
According to https://www.rockefeller.edu/fcrc/cellsorting/, I should use DPBS (Ca/Mg++ free) and DNAse when working with a sample with a lot of dead cells.
1. a PI stain relies on intercalating in DNA. If we use DNAse, we won't be able to visualize with PI. How do you optimize the buffer for single cell suspension when staining for DNA?
2. Should I use HBSS with Ca/Mg, or DPBS (Ca/Mg++ free), since I am working with cell death in lymphocyte cell line? In addition, an Annexin V kit (https://www.biolegend.com/en-gb/products/fitc-annexin-v-apoptosis-detection-kit-with-pi-8230) requires presence of calcium. Does using a Calcium free- PBS affect my staining?
Thank you!
DNAse can be helpful when your cell preparation is rich in nuclear debris and dead cells, as you correctly mentioned, getting rid of "sticky" extracellular DNA. You can use it with samples containing a a lot of polymorphonucleates, primary tumors and thawed samples with expected high cell death ratio which may affect sample quality for further applications. If you are using cell lines, I see no reason to treat your cell lines with DNAse during preparation before FACS, even if your experiment involves cell killing/death.
Using PBS w/o Ca/Mg and - even better - adding EDTA will help reducing cell death and clumping of difficult samples. This is especially important if sample preparation requires various steps and a long time before analysis (ie. isolating cells from mouse tissues). Keeping cells on ice will also help during long sample preparations.
PI and 7AAD are DNA intercalating vitality stains which will be totally fine with PBS+2%FBS+/- EDTA (or your favorite recipe) FC buffers when staining and washing your cells. They need to be added about 5-10 min before acquiring and do not require washing. I have used them in a sample previously treated with DNAase with no effect on the PI/7AAD stain, but I had washed the cells after DNAse and it was no present in the suspension used for analysis.
AnnexinV staining requires Ca+ ions to bind to negatively charged PS on cell membranes. Protocols usually involve staining your cells with your desired Ab mixture, washing and resuspending them in an "binding buffer" which usually contains CaCl and HEPES in order to bind AnnexinV to cell membranes.
Therefore, your combined workflow could look like this: prepare cells for staining (harvest, wash them and resuspend them in an appropriate volume - you could include a DNAse treatment here, if needed), stain them with your desired Ab mixture (temperature and time should be adjusted to your needs), wash them (with PBS+protein, ie. FBS2%), resuspend them in Annexin binding buffer (do not keep cells for long before analysis in this buffer), add conjugated AnnexinV and PI/7AAD, acquire your samples. If you have many samples or if you are FACS-sorting them, prepare them a few at a time, to avoid keeping them too long in Ann/binding buffer and with PI/7AAD.
Finally, pay great attention to whichever chemotherapeutic agent you are using in your in vitro experiments: many are fluorescent (all anthracyclines, ie. Doxorubicin, Daunorubicin, Idarubicin; Anthracenediones ie. Mitoxantrone; Chromomycins, etc.) and will stain your cells. Depending on the dose, time exposure and time to analysis they may be detected by FACS.
You can try PBS, I have used it in murine primary cells. just make sure that you use the control group also with PBS,
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