I am trying to dual stain live and dead cells (MCF7 and A375 cells). The problem is that after treatment of drug, the dead cells come into suspension and washed away after staining. Only few dead cells remains that are adherent but majority of dead cells in suspension are washed away during PBS washing. How can I troubleshoot this problem?
Ok, so I'm not entirely sure if you just want to measure the fraction of dead cells that are still attached or also the floating dead cells which you loose during washing.
If you want to measure the latter (floating) cells you can either take off the medium and spin them down and stain them separately (so you know how many floating cells you had), or you can mix them with the cells that are still adherent if you trypsinise them (basically combining floating and adherent cells). Flow cytometry staining would probably be a good start to be able to distinguish the two groups and there are many dyes around that are used for dead cell exclusion, e.g. PI (propidium iodide).
If you want to distinguish between dead cells that were still on the plate and dead floating cells in the sample you could maybe stain the cells from the plate separately with one marker (make sure it also stains dead cells), then combine them with the others and stain all for dead cells. Again, flow cytometry is probably your best bet here as you can do various analyses to see how many dead and live cells you have.
I hope this helps, good luck
Ok, so I'm not entirely sure if you just want to measure the fraction of dead cells that are still attached or also the floating dead cells which you loose during washing.
If you want to measure the latter (floating) cells you can either take off the medium and spin them down and stain them separately (so you know how many floating cells you had), or you can mix them with the cells that are still adherent if you trypsinise them (basically combining floating and adherent cells). Flow cytometry staining would probably be a good start to be able to distinguish the two groups and there are many dyes around that are used for dead cell exclusion, e.g. PI (propidium iodide).
If you want to distinguish between dead cells that were still on the plate and dead floating cells in the sample you could maybe stain the cells from the plate separately with one marker (make sure it also stains dead cells), then combine them with the others and stain all for dead cells. Again, flow cytometry is probably your best bet here as you can do various analyses to see how many dead and live cells you have.
I hope this helps, good luck
Best way is to do cytospin if you have cytospin centrifuge.
https://www.google.com/search?q=cytospin&biw=1225&bih=685&tbm=isch&tbo=u&source=univ&sa=X&ei=_dWfVcTnJMq5-AHN3YKgBQ&sqi=2&ved=0CCsQsAQ&dpr=1
Try this
http://www.lifetechnologies.com/us/en/home/brands/molecular-probes/key-molecular-probes-products/live-dead-viability-brand-page.html
Hello, you can use polylysin for surface pretreatment. This type of fixation is based on weak interactions of the charge, so the dead cells are not lost in the suspension, but to wash all still have to be careful.
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