Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(6)
Lymphocytes were prepared from two rat spleens minced in 10 ml aMEM (Gibco, Paisley, uK) containing 10% (v/v) fetal calf serum (FCS; Gibco, UK). Tissue pieces were disaggregated in a Potter homogenizer (four to five strokes). The cell suspension was centrifuged at 1000 X g for 3 min and the supernatant removed and centrifuged at 1500 X g for 5 min. The pellet was resuspended in 8 ml aMEM/FCS and divided into two equal aliquots each of which was layered onto 3 ml Ficoll-Paque (Pharmacia, Milton Keynes, UK). These were centrifuged at 1000 X g for 40 min, and the lymphocyte band (present at the interphase between the clear lower and cloudy upper layers) was collected and centrifuged at 1500 X g for 5 min. The resulting pellet, consisting of washed purified lymphocytes, was treated as described previously for spermatozoa.
Red blood cells were prepared by mixing 4 ml of whole blood with 55 |xl of acid dextrose citrate solution (403.8 mM NaCl, 16 mM NaH2PO4, 161 mM glucose, and 15.6 mM citrate). The suspension of red cells was then washed and treated as described previously for spermatozoa. ventolin-inhalers.com
Electrophoresis and Western Blotting
Proteins were separated by SDS-PAGE under nonreducing or reducing (by heating at 100°C for 5 min with 2 mM dithiothreitol [DTT]) conditions.