Looking for a stain that could be used on living cells to sort populations to identify higher (total or cytoplasmic) protein containing cells. Not looking for probes that bind specifically to certain proteins and the stain must work on living cells rather than fixed.
Hi Andrew,
The main problem I see in your question is that you want a proteinophilic dye (polar, charged) that can cross plasma membrane (apolar). If you use esterified derivatives of known polar probes, such as Fluorescein-Di-Acetate, you will not measure the amount of proteins, but the activity of endogenous esterases.
I am looking at Coomassie Brilliant Blue, which absorbs at 595 nm when bound to proteins, and, despite its huge molecule and the presence of some polar residues (sulfo, amino), the core molecule seems to be apolar, maybe enough to cross plasma membranes. It is worth trying, and Coomassie blue is relatively cheap. First under a confocal microscope with 561 or 633 excitation and IR emission, then try several concentrations of dye before analyzing with flow cytometer.
Oliv
Calcein-AM allows you to sort all living cells with very strong fluorescence at 488 excitation FITC channel. Doesn't harm the cells, don't change the expression profile if you want to culture the cells afterward over time it dissipates in about a week.
https://www.sigmaaldrich.com/catalog/product/sigma/17783?lang=en®ion=US
You might want to use 1 ug/ml and incubate for about 15-30 min before sorting/analysis. You can also combine it with other antibody markers i.e. all living cells at 488 channel + CD45 positive at PE channel.
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