Ethidium Monoazide (EMA)

Adapted from Kathrine A. Muirhead
Zynaxis Cell Science, Inc.

1. Use ethidium monoazide (EMA) that has been titered (0.5 ug/ml - 10 ug/ml) and compared to trypan blue exclusion and/or propidium iodide (PI= 0.5 ug/ml). Viable cells should have low background EMA staining.
2. Wash cells twice in phosphate buffered saline (PBS) containing 0.5% to 5% bovine serum albumin (BSA) and 0.1% sodium azide (NaN₃).
3. Resuspend cells to 10⁷ per ml.
4. Add 50 ul cells and appropriate volume of buffer to give 90 ul in 96-well plate or 12 x 75 mm tubes. Add sufficient EMA stock to each well or tube for the final concentration (as determined above) and mix well. (eg 10 ul of 100 ug/ml EMA in PBS).
5. Incubate IN THE DARK on ice for 15 minutes to allow uptake of EMA by dead cells.
6. Continue incubating ON ICE for 15 minutes 30 cm from fluorescenct light source to photoactivvate EMA and cause covalent binding.
7. Add appropriate amount of monoclonal antibodies and incubate 15 - 30 minutes on ice in the dark.
8. Wash twice with wash buffer (eg PBS-BSA-NaN₃).
9. Fix cells with 0.5% final concentration protein-free buffered paraformaldehyde (PFA) and store at 4°C IN DARK.

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Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility