Figure 1
Preparation and flow cytometric analysis of BODIPY-cholesterol labelled boar spermatozoa. (A) To prepare spermatozoa for the BODIPY-cholesterol assay, cells are first labelled with BODIPY-cholesterol in a non-capacitating media (1), then excess label is removed via density gradient centrifugation and spermatozoa can be incubated in various capacitating conditions (2). Following capacitation (2 h), spermatozoa can be analysed with flow cytometry and to determine cholesterol efflux, the percent loss in BODIPY-cholesterol fluorescence relative to the non-capacitating control (NC) was calculated (i.e. BODIPY-cholesterol fluorescence in TALP ÷ BODIPY-cholesterol fluorescence in NC × 100). (B) The resulting density plot of recorded sperm events with BODIPY-cholesterol and counterstained with propidium iodide (PI) to isolate a viable (PI−) and non-viable population (PI+) for analysis. (C) After selecting the viable population only, a loss in BODIPY-cholesterol can be observed following 2 h incubation in capacitating conditions (as indicated by black arrow), TALP and TALP supplemented with cAMP up-regulators (TALP+) when compared to the non-capacitating control (NC).
