Hi everyone,
I use a density gradient medium, Polymorphprep to separate lymphocytes and neutrophils from human whole blood. In the literature, I have found that people do try to culture them in medium post separation but a lot of these methods seemed to be quite tedious. I am wondering whether anyone has tried this and whether there are some quick and easy protocols to do this ?
I will be really grateful if you could share your experience.
~Ani
I have a suggestion about neutrophil isolation.
Neutrophil isolation (modified from Bryan Heit)
1. 20 ml of whole blood is mixed with 4 ml of ACD in a 50 ml conical plastic tube.
2. Mix 12 ml of 6% dextran with 24 ml of blood + ACD mixture. (for 6% dextran: 6 parts of Dextran 40 and 4 parts of 0.9% NaCl may use). Mix gently 18-20 times.
3. The mixture is transferred to 15 ml plastic tubes as four pieces. They are placed and kept vertically in the tube sports for 1-1.5 hours at room temperature. The duration can be extended to 2 hours.
4.The yellow supernatant is collected with a Pasteur pipetor and placed in a 50 ml tube. Centrifuge at 3000 rpm at 4 ° C for 15 minutes.
5. The supernatant is discarded. It is mixed with 12 ml of ice-cold distilled water for 30 seconds and mixed several times by adding 4 ml of 0.6M KCl. The solution is diluted with 50 ml of PBS and centrifuged at 4 ° C for 10 minutes at 2400 rpm.
6. Step 5 can be repeated 1-2 times until erythrocytes totally removed.
7. The pellet is diluted with 2.5 ml PBS.
8. Three ml of Ficoll is placed into the 15 ml tube and slowly put 2.5 ml of cell suspension. The tube is centrifuged at 1.600 rpm and 4 ° C, at 30 min without braking.
9. Buffy coat contains monocytes and lymphocytes. The pellet is made up of neutrophils. It is used by washing 2 times with PBS.
ACD:
7. 36 gr Citric Acid
14. 71 gr Sodium Citrate
9. 91 gr Dextrose is supplemented with 250 ml of double distilled water. It is stored at +4 C.
6% Dextran:
15. 00 gr Dextran (100 000 MW)
2. 25 gr NaCl is supplemented with 250 ml of double distilled water. It is stored at +4 C..
6M KCl:
11. 18 gm KCl is supplemented with 250 ml of double distilled water. It is stored at +4 C..
Best regards
After you get the lymphocytes and neutrophils in different tubes, count these.
A number of stimulation are used to see specific signalling in lymphocytes. As we do stimulation with PHA and IL2 for 72 hrs to see proliferation of mononuclear cells and PMA+ Ionomycin for 5 hrs (at optimized concentrations) for evaluate Th17, CD154, CD69, CD107a signalling.
what culture you need to do?
what outcome do you expect from culture?
There are no easy ways to separate leukocyte populations. And depending on what you want to do, the more pure of a population you want, the more stringent the separation techniques will be.