Hi All,
I am having a problem with pelleting down the neutrophils after isolation.
I use 300g for 10minutes, but I seem to be loosing most of the cells.
Can anybody recommend what I can do to improve my yield?
Is there a type of cells can I use to "pull them" down better?
Any recommendations are highly appreciated
Thanks
300g should not be problematic. I have seen many protocols that use 350g. Make sure to use very low acceleration and turn the brake off. I would also suggest being more careful with your pipetting and vortexing. The shear stress during these activities can be far greater than the that of centrifuging at 300g.
Hi Aleks,
I agree with Ron, 300g should be more than sufficient to spin down neutrophils, and it is a good advice to keep the brakes off as Ron suggested. If you are using Polymorphprep (or similar techniques) to isolate neutrophils, make sure to suspend the neutrophils in a large volume of PBS/medium in the first couple of washes and carefully mix the neutrophils before spinning them down. Otherwise residual density gradient could block neutrophils from pelleting efficiently.
Good luck!
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology