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IACF-Flow Cytometry Facility- Live-Dead Discrimination and Cell Counting

Cell Counting/ Live-Dead Discrimination

This is a microscopy based application for definitive discrimination of live and dead cells. Trypan Blue exclusion notoriously over estimates the number of dead cells. Using this protocol, you can obtain a more accurate count of cells.

Stock solution
Prepare 3mg/ml ethidium bromide in absolute ethanol.
Prepare Acridine Orange at 5mg/ml in absolute ethanol.

Store this stock in a dark vial, refrigerated.

Working solution

Take 1ul of each added to 1ul of PBS. This can be stored at RT and can be made every couple of weeks.
Dilute cells with an equal volume of the working solution and immediately look on the fluorescent microscope.
Dilute cells if cell count is to high.
Remember to take this into account when you calculate original numbers

Green is Live, Red is Dead

Note: these chemicals are highly carcinogenic.
PI can be used in place of EB at the same concentration