反应详情

Neutrophil adhesion: Isolated neutrophils were resuspended in M1 99 containing 1% BSA with a final concentration of 3×106 cells per ml. Cells suspensions (1 ml) were incubated with or without a test compound, 16(S)-HETE, 16(R)-HETE, or 20-HETE (concentrations 0.01 to 10 μM) for 10 min and then placed into gelatin-coated wells and further incubated for 20 min at 37° C. The wells were washed three times with 0.5 ml of solution M199 to remove non-adherent cells. The adherent cells were then removed by addition of 0.4 ml of 0.25% trypsin-EDTA in 0.1 M PBS to the gelatin-coated wells and incubation for 10 min at 37° C. The incubates were transferred and the wells were washed with 0.1 ml of M199 solution. The amount of cells in combined solutions was counted. In some experiments, the adhesion of neutrophils was stimulated by addition of 1 U/ml of thrombin to neutrophil suspension immediately prior to the transfer of cells to the wells. Experiments were performed in duplicates for each concentration of HETE. The inhibition of adhesion was expressed as percent of cells remaining in suspension (non-adherent) relative to control. The range of adherent PMNs in control incubations was 6 to 23%. This was increased two-fold in the presence of thrombin.

1. customNeutrophil adhesion: Isolated neutrophils
2. customfor 10 min
3. washThe wells were washed three times with 0.5 ml of solution M199
4. customto remove non-adherent cells
5. customThe adherent cells were then removed by addition of 0.4 ml of 0.25% trypsin-EDTA in 0.1 M PBS to the gelatin-coated wells and incubation for 10 min at 37° C
6. washthe wells were washed with 0.1 ml of M199 solution
7. additionIn some experiments, the adhesion of neutrophils was stimulated by addition of 1 U/ml of thrombin to neutrophil suspension immediately
8. temperatureThis was increased two-fold in the presence of thrombin