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Treat semen samples within 8 hours after ejaculation. Store semen samples at room temperature.
Check the sperm count using a light microscope. Dilute the semen 1:1 or 1:9 with PBS according to the sperm count (the greater the count the greater the dilution). Sperm and leukocyte count
Sperm and leukocyte count: Visualize measured data as a dot-plot, where FITC fluorescence intensity is on the X-axis and forward-scatter (FSC) is on the Y-axis. Set gates: gate A for all cells, gate B for sperms and gate C for Fluorescent Count Standard (fluorescent beads). Then visualize events from the gate A as a dot-plot, where the X-axis represents fluorescence intensity of PE-Cy™5 dye and the Y-axis represents side-scatter (SSC). On the contrary to the sperms, leukocytes are CD45 positive. Separate the bright population of leukocytes using gate D. Calculate sperm and leukocyte count using following formulas: S = (gateB/gateC) x dilution [106 sperm/mL] L = (gateD/gateC) x dilution [106 leukocytes/mL] S represents sperm count in ejaculate L represents leukocyte count in ejaculate Gate B represents event count in gate B Gate C represents event count in gate C Gate D represents event count in gate D Sperm viability: Visualize measured data as a forward-scatter (FSC) versus side-scatter (SSC) dot-plot. Set the gate around sperm cells. Then visualize sperm cells in a histogram , where the X-axis represents fluorescence intensity of propidium iodide in PE-Cy5 or PerCP channel. Separate positive and negative sperm cells using appropriate gate. Negative population represents viable sperms, while PI-positive population represents non-viable sperms. Viability of sperms is represented by the percentage of viable sperms from all sperm cells. Acrosome integrity and presence of intra-acrosomal protein: Visualize permeabilized and non-permeabilized samples stained using antibody against intra-acrosomal protein as a forward-scatter (FSC) versus side-scatter (SSC) dot-plot and set the gate around sperm events. Then visualize sperm events in a histogram, where the X-axis represents fluorescence intensity in FITC fluorescence detector. Separate positive and negative sperms using appropriate gates. Expected values: Normal values according to WHO :