Improved evaluation of mitochondrial membrane potential in bovine spermatozoa using JC-1 with flow cytometry

Abstract One of the most popular methods for measuring mitochondrial membrane potential (MMP) via conventional flow cytometry in spermatozoa is through the use of the fluorescent dye 5,5,6,6′-tetrachloro-1,1′,3,3’tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). The JC-1 dye is unique in that it will fluoresce green (525 nm, monomers) or red-orange (595 nm, J-aggregates) when excited to indicate depolarized or polarized mitochondria. While JC-1 can be multiplexed with viability dyes such as propidium iodide and SYBR 14 for microscopy, doing so for conventional flow cytometry has proved difficult due to overlap of the emission spectra between dyes. The objective of this protocol is to improve the accuracy of JC1 J-aggregate quantification in conventional flow cytometry by using a 405 nm or 532 nm laser for excitation coupled with the viability dye calcein violet (CV). Quantification of live J-aggregates when excited with a 405 nm or 532 nm laser is more strongly correlated to progressive motility (405 nm: r = 0.73, P < 0.0001; 532 nm: r = 0.52, P = 0.0002) and viability (405 nm: r = 0.93, P < 0.0001; 532 nm: r = 0.74, P < 0.0001) than quantification with the traditional 488 nm laser excitation and dual emission of 525 nm and 595 nm (progressive motility: r = 0.05, P = 0.72; viability: r = 0.21, P = 0.14). The use of a 405 nm or a 532 nm laser requires no compensation. This allows for clear identification of the polarized J-aggregates and, when combined with CV, may help identify which spermatozoa have the greatest fertilization potential.