Recently, I purify murine bone marrow neutrophil through 3-layer Percoll Gradients recently(72%-62%-56%), the purity is so low, between 40%-60%. I always follow the following steps:1. Grind the tibias and femurs in HBSS, filter the cell suspension with 40um strainer, wash 3 times; 2. Layer the cell suspension on 56% Percoll, centrifuge at 1500g for 30min with no break at RT; 3.Discard the frist two layers and harvest the third layer between 62% and 72% into RPMI; 4. Wash with RPMI 3 times. 5. Double stain CD11b and Ly6G to detect the purity.
I did not lysed red blood cells, is this step necessary? The cell layer is clear, as the picture showed, and I am very careful when harvest the neutrophils, but the number of neutrophil I got is always 1.5 to 2 times than other protocol mentioned, at such a low purity. Can anyone give me suggestion to got higher purity?
Hi Zhimin,
There are several ways to get purified PMN population at the step where you actually stuck. Re-suspend the cell suspension in HBSS or suitable buffer and layer it on Histopaque 1077-1119 gradient. This will give you pure population of BMD PMNs. I personally have good experience with Histopaque 1077-1119 density gradient method. But one can also repeat the percoll step with 62-72% gradient. I think this may solve your problem. Make sure you should have single cell suspension before layering. Try to eliminate RBCs before moving toward FACS.
Best Luck
Durgesh
Were you have to increased the efficiency? I'm trying to do the same and cannot seem to go above 60-70% purity for neutrophils.
Hi Christine,
Have you check the purity with flow cytometry? If yes, would you please share FSC Vs SSC profile. Then we can easily pinpoint the exact contaminant population. I believe that you might have RBC or platelet as a contaminant in the purified neutrophils.
Try granulosep or gradient of histopaque1077 and 1119 to enhance purity of your neutrophil population.
Yes, I have. That's how I have been checking for purity after histopqaue.
Attached is an image of before/ after isolation. I have also attached an image of the tube after centrifugation.
HI Christine Youn
Have you solve the problem in neutrophil isolation？I am also facing with the low purity using 81% and 63% Percoll gradients. I will be greatful if you can share your experience.
- Badges
- Science method