Washed sperm cells were also incubated in the absence of biotinylated carbohydrate conjugates with 100 |xl of streptavidin-FITC (negative control). The specimens were counterstained for cell viability using propidium iodide and only propidium iodide-negative cells (living cells) were imaged (single scans were made to freeze motile cells). Staining of the sperm was visualized using an inverted spectral emission con-focal laser-scanning microscope (CLSM) (Leica TSC-SP GmbH, Heidelberg, Germany) or alternatively analyzed by flow cytometry. Flow cytometric analysis was performed on a FACScan flow cytometer equipped with a 100-mW argon laser excitation at 488 nm (Becton Dickinson, San Jose, CA).
Five minutes before analysis, sperm cells were counterstained with propidium iodide for simultaneous detection of carbohydrate binding and viability status of individual sperm cells. Cell deterioration was minimal and deteriorated cells show similar FITC fluorescence as positive stained cells (data not shown). After gating out nonsperm and aggregated events (on FSC and SSC scatter properties) and deteriorated cells (on FL-3 fluorescence properties; 630 nm long-pass filter), fluorescein-conjugated carbohydrates bound to the live sperm cells were quantitated on the FL-1 detector (530/30-nm band-pass filter).