Easykit 1 viability and concentration

Plasma membrane integrity was evaluated using the EasyKit 1 Viability and Concentration (ref. 024708; IMV Technologies) according to the manufacturer’s instructions and as previously reported [33 (link)]. The kit is based on two fluorescent probes: SYBR and propidium iodide (PI). SYBR stains nucleic acid and labels all spermatozoon heads green, whereas PI penetrates only membrane-damaged spermatozoa and labels spermatozoon heads red (Fig 2A). From each sample, 2 ΔL of homogeneous spermatozoa at 57 x 10⁶/mL was added into the well of a 96-well plate containing 199 ΔL EasyBuffer B (ref. 023826; IMV Technologies). The contents of each well were homogenized by pipetting, and the plate was covered and placed in an oven at 38.5°C, and protected from light for 10 min. A total of 5000 spermatozoa were counted in the flow cytometry reading. The results are expressed as the percentage of viable spermatozoa.

Plasma membrane integrity was evaluated using the EasyKit 1 Viability and Concentration (ref. 024708; IMV Technologies) according to the manufacturer’s instructions and as previously reported [95 ,96 (link)]. The kit contains two fluorochromes and allows one to distinguish between live and dead spermatozoa. From each sample, 2 µL of homogeneous spermatozoa at 57 × 106/mL was added to the well of a 96-well plate containing 199 µL EasyBuffer B (ref. 023826; IMV Technologies). The contents of each well were homogenized by pipetting, and the plate was covered and placed in an oven at 38.5 °C and protected from light for 10 min. A total of 5000 spermatozoa were counted in the flow cytometry reading. The results are expressed as the percentage of viable spermatozoa.