I tried to isolate neutrophil from human venous blood followed the protocol as below:

1. Mix heparinized blood sample (2ml) with 2ml 3% dextran in syringe, leave it for 30 min. at room temp.

2. Directly transfer upper layer from syringe onto ficoll layer (with the half volume)

3. centrifuge at 400 g for 40 min at room temp.

4. Aspirate and discard supernatant above the PMN-erythrocyte layer

5. Resuspend the plet and then add 1 ml H2O and mix well for 28 s

6. Add 5 ml of PBS and mix

7. Centrifuge at 500g for 5 min at 4°C.

8. Discard supernatant and repead this step (only once) if more lysis is needed.

9. Discard supernatant and resuspend the pelet in RPMI 1640 containing 10% FBS.

we always see neutrophils clumping either at step 5 or at the end of the process. We dont know the reason.