In processing rat spleen for preparation of single cell suspensions, we seem to be having problems with precipitation of material after adding the lysis buffer. Spleen is collected fresh and immediately placed into ice cold HBSS w/o Ca, Mg, phenol red but with or without 2%FBS and 1micromolar EDTA. Spleen is cut into small pieces and gently pressed through a wire screen. The cell mix is then passed through 70 micron nylon. The cells are spun for 10 minutes at 4C at 250 g, the pellet is dislodged and the cells are brought up in 5 mL of standard ammonium chloride lysis buffer from Sigma. All steps are on ice. At this point precipitated material immediately forms. Any advice?
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