Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(7)
Membrane Integrity Studies
The percentages of sperm with an intact acrosome were evaluated by fluorescence microscopy (Olympus BX-60 model; Olympus Corp., Lake Success, NY) after fixation and ethanol permeabilization of the sperm pellets and subsequent staining with fluorescein (FITC)-conjugated Pisum sativum lectin (Sigma). In positive control sperm, the ac-rosome reaction was induced by incubating (3 h at 37°C) the sperm suspension with 10 ^M calcium ionophore (CaI) A23187 (Sigma). Motile sperm (5 X 106/ml) were incubated for 4 h at 37°C in the presence and absence of 100 |xM each of WHI-01, WHI-03, WHI-05, WHI-07, or N-9 followed by the addition of propidium iodide (PI; 10 ^g/ ml) and analyzed by flow cytometry. buy diabetes drugs
The percentage of sperm positive for PI was determined by flow cytometry using a fluorescence-activated cell sorting Vantage flow cy-tometer (Becton Dickinson and Co., Mountain View, CA). The sperm in the swim-up fractions were identified by their characteristic forward and 90° angle light-scattering properties. All analyses were done using the 488-nm excitation from an krypton/argon laser with a 635-nm band pass filter for PI emission. The percentage of sperm positive for PI staining was determined using cutoff signals for membrane-intact motile sperm.