I will use MCF-7 cell line, so after treatment and incubation, do I need to remove media, wash with PBS, add trypsin and new media then take a sample and mix with trypan blue? and how much uL of each reagent needed to do so?
Hello Reem,
It is certainly possible but you need to wash wells a couple of times with PBS to get rid of serum proteins towards which Trypan Blue has strong affinity. Secondly, after adding Trypan Blue to the cells, you need to fix the cells so that prolonged incubation with Trypan Blue does not give you false positive results.
Briefly, after washing, add 0.2% Trypan Blue solution (0.4% Trypan Blue stock can be diluted 1:1 with PBS) for 1 min. Remove the Trypan Blue solution and immediately fix with 4% paraformaldehyde, pH 7.5, for 10 min at room temp. Rinse 3–4 times with PBS with gentle shaking until PBS is no longer blue. Count the cells.
Dear Reem,
I would think that it would be alot easier to grow your cells on coverslips placed in 24-well or 48-well plates and then remove the coverslips containing cells with sharp forceps when you want to do so without any tripsin or chemical treatment.
best wishes,
Refik
Thanks for advice.But unfortunately I have only 96-well plates and I have limited time to start the experiments with the available resources.
Trypan Blue is pretty simple to use - don't overthink it too much. A 1:20 dilution of 0.4% stock, gently swirled, will be good to identify dead cells in a 96-well plate by brightfield microscopy. Even if you get close, but not exactly there, it will be fine. Trypan Blue is fairly binary (positive staining dead, non-positive alive). So just get a few microliters into a well and you will be okay.
Hello Reem,
It is certainly possible but you need to wash wells a couple of times with PBS to get rid of serum proteins towards which Trypan Blue has strong affinity. Secondly, after adding Trypan Blue to the cells, you need to fix the cells so that prolonged incubation with Trypan Blue does not give you false positive results.
Briefly, after washing, add 0.2% Trypan Blue solution (0.4% Trypan Blue stock can be diluted 1:1 with PBS) for 1 min. Remove the Trypan Blue solution and immediately fix with 4% paraformaldehyde, pH 7.5, for 10 min at room temp. Rinse 3–4 times with PBS with gentle shaking until PBS is no longer blue. Count the cells.
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