Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(5)


Quantitation of 2D6 McAb Binding to Spermatozoa by Fluorescence-Activated Cell Sorting (FACS)

The amount of 2D6 McAb bound to spermatozoa was quantified by FACS analysis using a Beckton Dickinson 2 laser FACScalibur, and the resulting data were analyzed on Cell Quest software (Beckton Dickinson, Franklin Lakes, NJ). Spermatozoa from the specified epididymal region were washed twice in PBS/1 mM Pefabloc as described previously and resuspended in PBS/0.1% (w/v) BSA. The final washed sperm pellet was then treated as follows: (A) Resuspension in 150 |xl of 2D6 McAb supernatant plus 50 |xl PBS/0.1% (w/v) BSA for 1 h with gentle rotation, washed twice as previously described, and incubated in 100 |xl FITC-rabbit anti-mouse IgG (diluted 1:20 in PBS/0.1% [w/v] BSA).

Sperm were washed twice more in PBS/0.1% (w/v) BSA and analyzed by FaCS. (B) Resuspension in 500 |xl dilute caudal plasma (CEP; protein concentration 1 mg/ml) for 60 min at room temperature, washed, and treated as described in (A). In some experiments, CEP was dialyzed against PBS/1 mM Pe-fabloc at 4°C for 48 h. Prior to FACS analysis, samples were diluted to ~5 X 104 sperm/ml in PBS/BSA, and the instrument was operated at a flow rate of 100-500 cells/sec. ‘‘Gating’’ was used to remove cell clumps and debris from the data analyzed. Gates were set using washed caudal sperm (no first layer antibody) as a control. This analysis ignored signals with very low forward and sideways scatter (predominantly debris) or high forward scatter (frequently due to clumped cells or aggregated debris).

Category: Plasma

Tags: epididymis, gamete biology, male reproductive tract, sperm