Do not increase the volumes of blood or paraformaldehyde in the tube, as this will increase the possibility of platelet aggregation. Fixed platelets are stable up to 5 days. Resuspend the pellet by vortexing thoroughly. Label clean 12 x 75-mm test tubes, and add 20 L of monoclonal antibody to the appropriately labeled test tubes. Using a fresh micropipettor tip each time, carefully add 50 L of the fixed blood suspension to the bottom of each tube.
Exercise care to prevent blood from running down the side of the tube.
If the blood remains on the side of the tube, it may not be stained with the reagent. Protect samples from light during this procedure. Centrifuge at 1200 x g for 5 minutes at room temperature, and aspirate supernatant. If samples will not be analyzed within 3 hours, proceed to step 9, paraformaldehyde fixation. BD recommends that samples be analyzed within 3 hours.