Hoechst 33342 h342

The fraction of acrosome‐intact spermatozoa was determined as described in detail before.¹⁹ Briefly, aliquots of 3 × 10⁶ cells were washed twice in Dulbecco's phosphate‐buffered saline (PBS, ThermoFischer scientific) (500×g, 10 min), and the pellet re‐suspended in a staining solution containing (final concentrations): 5 µg/ml fluorescein isothiocyanate conjugated Pisum Sativum agglutinin (FITC‐PSA, Sigma–Aldrich, MO, USA), 0.5 µl/ml propidium iodide (PI, ChemoMetec A/S, Allerød, Denmark) and 10 µl/ml Hoechst‐33342 (H342, ChemoMetec A/S, Allerød, Denmark) in PBS. The samples were incubated at 37°C for 30 min on a shaker, followed by mixing with 100 µl of an immobilising solution containing 0.6 M NaHCO₃ and 0.37% (v/v) formaldehyde in distilled water. The samples were loaded onto two‐chamber NC‐Slide A2™ slides (ChemoMetec) and analysed by image cytometry using a NucleoCounter® NC‐3000™ (ChemoMetec) and a FlexiCyte protocol. The PI intensity was plotted as a function of FITC‐PSA intensity on bi‐exponential scales, and specific quadrant gates, as published before,¹⁹ were used to identify the fraction of acrosome‐intact viable spermatozoa.