Anti cd45 clone d058 1283

Seminal cells were separated from seminal plasma immediately after collection by centrifugation for 15 min at 775 x g, resuspended in 1 ml cold PBS supplemented with 10% FCS and 2 mM EDTA, and kept on ice for no more than 1 h. Cells were then centrifuged for 10 min at 1500 x g, filtered through a 70-µM sieve, and washed with 5 ml cold PBS supplemented with 10% FCS and 2 mM EDTA.
CD45⁺ semen cells from animals #BA865F, #1103075, #CA147F, and #MF1414 were enriched using CD45 magnetic beads and cell sorting with a BD FACS Aria Flow Cytometer. Total semen cells were incubated for 15 min at 4 °C with 20 μl anti-CD45 magnetic beads (Miltenyi Biotec) and washed once with 2 ml cold PBS supplemented with 0.5% BSA and 2 mM EDTA (sorting buffer). The CD45⁺ cell fraction was then enriched by magnetic-bead sorting, using LS columns (Miltenyi Biotec), according to the manufacturer's instructions. Cells were eluted in 4 ml sorting buffer. Following the magnetic bead-based enrichment process, cells were stained with amine-reactive blue dye (Life Technologies) to identify the dead cells, and anti-CD45 (clone D058-1283, BD Pharmingen). Cells were washed twice and stored at 4 °C in PBS/10% FCS. CD45⁺ cells were then sorted by simultaneous four-way sorting on a FACS Aria flow cytometer (BD Biosciences). The enriched cell fraction was used in the neutralization assay.