The fixed PMNs are separated from the red blood cells in a subsequent step. The F-actin staining and the evaluation follow established methods. The method is fast, easy to handle, inexpensive, and yields well-reproducible results. Vacuum systems are not suitable since they activate the PMNs. After 20 s, PMN activation was stopped by fixation.
Glycerol addition lowers the freezing point and accelerates red blood cell lysis.
The supernatants were discarded. The pellets were resuspended in 1 ml of distilled water, and the red blood cells were lysed at room temperature for 20 min, centrifuged for 5 min at 200 g , and the supernatants containing the remnants of the red blood cells were discarded. To prevent impairment of erythrocyte lysis, the whole fixation time should not exceed 20 min. After thawing, the samples can be processed. Since the fixation time influences FITC-phalloidin binding, fixation conditions should be constant.