Isolation of Monocytes FGB

Revised 8/27/96

1. Get petri dishes containing monocytes from 37'C incubator.

2. Do forcible pipetting and collect the cells in the medium one dish at a time.

3. Quickly add 10ml of cold HBSS (Ca-, Mg-), to the dish you're working with.

4. Incubate at 4'C for 30 minutes. Repeat with other dishes.

5. One dish at a time, harvest cells again by forcible pipetting 5-6 times all over the petri dish.

6. Collect supernatant into a 50ml conical tube then add 10ml of fresh, cold HBSS this time to first dish only.

7. Be sure to keep the conical tube with cells on ice when not in use.

8. Use the10ml from the first dish to sequentially wash all dishes.

9. Collect the 10ml at end and add to previous tube.

10. Keep the cells on ice until ready to spin.

11. Spin cells for 10 minutes/4'C/1200 rpm.

12. Aspirate supernatant then pool if possible.

13. Add 10-20ml RPMI 4+ to wash, spin for 10 min/4'C/1000 rpm.

14. Repeat steps 12-13.

Now the cells are ready to be frozen or used the same day.

To freeze cells see Freezing cells protocol or resuspend pellet in an appropriate volume of RPMI 4+ for same day use.