I want to isolate human neutrophil after getting the PBMC first.
I first put the diluted blood (2ml blood with 2 ml PBS) onto the ficoll paque to extract PBMC.
The I took out the bottom red portion, added 5ml PBS for dilution, and then mixed with the 8ml solution that consists of 3% dextran and 0.9% NaCl. After standing for 30mins, I got the uppermost layer of solution (pink in color) which was supposed to have neutrophil.
However, I found that it seems I don't have neutrophil after centrifugation; there are red clumps in the bottom, I think they are only red cells.
Is there anything wrong in the protocol I used? Or anymore can suggest an alternative protocol?
What should the color of the neutrophil supernatant be? Should it be pink? How many supernatants I should get?
Hello,
Washing with PBS1X a couple of times works when I extract splenocytes. I get a white pellet.
About neutrophils, I think maybe you could try to incubate first with dextran and NaCl, then washing with PBS1X, and finally incubate with ficoll.
This article might help: Boyum A. "Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 19." Scand J Clinc Invest 21 (Suppl 97): 77 - 89, 1968
Good luck
We have used HetaSep from StemCell Technologies. It contains Hetastarch which causes aggregation of RBCs and you do not lose the granulocytes to the lower fraction (All nucleated cells are found in the upper fraction). If you wash with PBS 2x and layer on Ficoll you will get great separation of neutrophils to the lower fraction of the Ficoll. According to
Another simple method is using a hemolysis buffer (150mM NH4Cl 10mM NaHCO3 0.1mM EDTA) after ficoll.
You could use dextran sedimentation followed by discontinuous plasma-Percol gradient centrifugation which would allow you to take off both the PBMCs and neutrophils from different layers simultaneously. Also gives you minimally/unprimed neutrophils.
I agree with Hilton that you can just hemolyse rbcs after ficoll to get the granulocytes. I use ice cold 0,2% NaCl and then reconstitute with 1,6% NaCl, spin the cells down and then if there still are some red cells repeat the same thing. This is a very gentle method and you will get no unspecific stimulation of your cells. I you want pure neutrophils however, you have to consider that eosinophils will be mixed with neutrophils after ficoll centrifugation. They can be removed by magnetic sorting (with CCR3 magnetic beads for example).
I agree with Maria, but I use a different concentration of NaCl, first 0.2%, just for few minutes, because it can lyses the neutrophils too if you let for long periods, next 1.6% of NaCl.
I agree with Elena . Additionally my advise will be dextran and the incubation time.
1. You should use citrated blood.
2. Firstly, incubate with 6% dextran at room temperature for more one hour.
3. Collect to the upper part and applicate this part to Ficoll.
4. Remove PBMC layer and all from upper part.
5.Take just the bottom part.
6. Use that the Method recommended by Maria Lampinen for the hemolysis.
7.After washing with PB you recover more then 98% Neutrophils.
Good Luck
Dear Chan,
You can use 12 ml dextran 6% and 24 ml blood+anticoagulant (20 ml blood+4 ml ACD).
Best regards,
Ishak
Hi,
Please Find the following protocol.
PROCEDURE FOLLOWED IN 4 degree Celsius).
1. Load venous blood on Ficoll Paque Plus (GE Health care).
2. Remove all the layer by except RBC pellet gently.
3. Re-suspend RBC pellet (e.g. 5 ml) in 30 ml HBSS without calcium magnesium.
4. Centrifuge at 250 RCF for 10 minute (zero break and zero acceleration).
5. Remove supernatant HBSS without disturbing the pellet.
6. Perform water lysis for destroying RBCs. (Add 45 ml of autoclaved water into RBC pellet, followed by add 5 ml of 10X PBS. (This procedure should be as quick as possible).
7. Centrifuge above mixture at 350 RCF for 5 minutes.
8. Repeat this process one more time in order to completely eliminate RBS. (Water lysis should not be more than 3 times).
9. Mix the pellet gently. (Do not use pipette for mixing in any step of isolation).
10. Add 15 ml of HBSS Without Calcium and Magnesium and gently mix the pellet.
11. Centrifuge at 350 RCF for 5 minutes.
12. Remove supernatant and add RPMI1640+10% FBS in it.
13. Culture cell with density of 10^5 to 10^6 cells per milliliter.
14. Confirm it by Giemsa staining/ DAPI staining.
This is the most simplest method I regularly practiced in my Lab.
Try with this method.
Best regard!
Durgesh
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