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An equal volume of dye Mastermix (1 M JC-1, 0.005% digitonin, 10 g ul 1 oligomycin, 5 mM -mercaptoethanol in DTEP buffer) was added, and after 10 min at room temperature, the cells were added on top of the peptide template at a concentration of 40,000 cells per well

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The most critical temperature consideration is storing the reconstituted peptide in the refrigerator afterward