I want to measure cell death induced by a drug on Leishmania promastigotes by flow citometry. I thought of using PI staining to distinguish dead from live cells after treatment. The problem is, I cannot use live parasites in the flow citometry equipment because of biosafety concerns, so I need to kill the parasites to enter the facility. I though of staining with PI first and then fixing with PFA. The problem is, I am not sure if the PFA will make the cells membrane permeable or not. If it does, all the cells will incorporate PI and appear dead... Any ideas on how to solve this issue?
Dear Carrer, fixing the Leishmania parasite with paraformaldehyde (PFA) will not permeabilize the cell membrane as PFA causes covalent cross-links between molecules to form an insoluble network. PFA is used prior to permeabilization to keep the molecules intact. Just fix the parasites with 4% PFA and stain them with PI and PI will only stain dead parasites. As the parasites will not move/non-mobile, there will not be biosafety concerns. Just make sure to run decontamination cleaning in the Flow cytometer after use.
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