Mitochondrial transmembrane potential AWm can be estimated by cationic lipophilic dyes with high binding affinity to the negatively charged inner mitochondrial membrane (9,71,72). Because binding characteristics do not completely overlap, parallel staining with several dyes is recommended.
1. Resuspend 2 x 105 cells in 200 ^L of annexin binding buffer if concurrently stained with annexin V-FITC or annexin V-PE matching a potentiometric dye emitting FL-2 or FL-1 fluorescence, respectively. Alternatively, 2 x 105 cells can be resuspended in 200 ^L 5 mM HEPES-buffered saline (HBS; containing 0.9% NaCl at pH 7.4). This buffer, lacking Ca2+, does not allow concurrent staining with annexin V.
2. Aliquots of cell suspensions are stained with several potentiometric dyes in parallel.
a. Add 200 ^L of dye solution containing 20 nM DiOC6 (excitation 488 nm, emission 525 nm recorded in FL-1). This dye can be added in combination with annexin V-PE emitting FL-2 fluorescence.
b. Add 200 ^L of dye solution containing 1 ^M TMRM (excitation 549 nm, emission 573 nm recorded in FL-2). This dye can be added in combination with annexin V-FITC emitting FL-1 fluorescence.
c. Add 200 ^L of dye solution containing 0.5 ^M JC-1. JC-1 selectively incorporates into mitochondria, where it forms monomers (fluorescence in green, 527 nm) or aggregates at high transmembrane potentials (fluorescence in red, 590 nm) (73,74).
3. Parallel cell suspensions should be treated with 5 ^M mClCCP. Cotreatment with this protonophore for 15 min at 37°C results in decreased DiOC6, TMRM, and JC-1 fluorescence and serves as a positive control for disruption of mitochondrial transmembrane potential (15).
4. Incubate cells in the dark for 15 min at 37°C before flow cytometry.
5. For each sample, measurements are carried out on 10,000 cells.
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