Reagents:

• Ficoll-Paque Plus, Pharmacia (cat.no. 17-1440-02).

• Phosphate buffered Saline (PBS), BioWhittaker (cat.no. 17-515F).

• Tri Natrium Citrate Dihydrate (TNC), 38g/l.

• ACK lysing buffer, BioWhittaker (cat.no. 10-548E).

• peripheral blood sample (e.g. 50mls of heparinized blood)

Protocol:

1. Transfer blood to 50 ml tube.

2. Spin at 2000 rpm for 10 minutes.

3. Collect plasma and spin at 3000 rpm for 10 minutes.

4. Collect plasma and store at -80 °C.

5. Dilute remaining cell suspension with PBS/10%TNC to 50 ml.

6. Fill two 50 ml tubes with 12.5 ml Ficoll-Paque Plus each.

7. Gently pipette 25 ml of the diluted cell suspension on top of each 12.5 ml of Ficoll-Paque Plus.

8. Spin 20 minutes at 2000 rpm at room temperature. No brake!

9. Transfer the white blood cell ring fraction to a new 50 ml tube using sterile Pasteur's pipette.

10. Adjust the volume to 50 ml using PBS/10%TNC.

11. Spin 10 minutes at 1700 rpm.

12. Discard the supernatant.

13. Resuspend each pellet in 2 ml ACK lysing buffer to lyse remaining erythrocytes.

14. Incubate for no more than 2 minutes at room temperature.

15. Adjust the volume to 50 ml using PBS/10%TNC.

16. Spin 10 minutes at 1200 rpm.

17. Resuspend the pellet and count the cells.

Notes: Processing the blood this way will remove thrombocytes from the plasma without activating them, so that plasma can be used for testing of chemokine levels. Before starting Ficoll-Paque Plus and PBS with 10%TNC (v/v) should be at room temparature.

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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