This method is used to purify neutrophils from whole blood. It will render 2-4x106 neutrophils per ml of blood.
- Heparin (1,000 U/ml, Leo Pharmaceutical Products, Ballerup, Denmark)
- 50-ml polypropylene tubes
- Sterile disposable 5- and 10-ml plastic pipettes
- Sterile 21G needles (Becton Dickinson) and 5- and 10-ml syringes
- Adjustable 20-µl, 200-µl and 1000-µl pipettes and sterile disposable tips
- Sterile disposable 1.5- and 5-ml tubes
- Sterile distilled water
- 3.5% (w/v) sodium chloride
- Trypan blue (Sigma Chemical CO. St. Louis, U.S.A.)
- Ficoll (Lymphocyte separation medium, Gibco, BRL, Life Technologies Ltd, Paisley, Scotland)
- Hank's Balanced Salt Solution, without phenol red, calcium or magnesium
- (HBSS- Gibco, BRL, Life Technologies Ltd, Paisley, Scotland)
- Dextran T500 (Pharmacia Biotech AB, Uppsala, Sweden) (3% solution in HBSS-)
1. Draw venous blood in a sterile disposable syringe containing approximately 10 U of heparin per 1 ml of blood.
2. Transfer blood to a 50-ml sterile disposable tube.
3. Using a sterile 10-ml pipette add 1/2 volume Dextran T500 (3 % solution). Do not produce bubbles as this will damage blood cells and retard the settling of erythrocytes.
4. Allow the blood to settle for a maximum of 20 minutes. After this time the majority of erythrocytes will have settled to the bottom of the 50-ml tube (formation of two layers in the tube). The upper layer contains almost all white blood cells.
5. Transfer the top layer to a new 50-ml tube using a sterile disposable pipette followed by Ficoll (11 ml). Extra care should be taken when adding Ficoll, insert the pipette quickly into the solution with the tip touching the bottom of the tube. Slowly release 1 ml of Ficoll into the very bottom of the tube raise the pipette tip 1 cm up the side of the tube and slowly release 9 ml of Ficoll and discard the remaining 1 ml of Ficoll. Do not mix the two solutions. Slowly remove the pipette. A clear separation surface should exist between Ficoll and supernatant. MOVE EXTREMELY CAREFULLY!
6. Centrifuge at 1,500 rpm and 4oC for 20 minutes. After centrifugation the plasma, buffy layer and Ficoll should have formed distinct layers in that order and the neutrophils and remaining erythrocytes will be present in a pellet.
7. Lyse the erythrocytes by adding 5.8 ml of sterile water forcefully by a syringe, swirl continuously for a maximum of 20 seconds and immediately add 2 ml of 3.5 % (w/v) sodium chloride to stop lysis of neutrophils. Add up to 40 ml HBSS- and centrifuge at 1,500 rpm for 10 minutes.
8. Remove the supernatant and resuspend in about 1 ml HBSS-.
9. Quantify the total neutrophil number per ml as follows:
To 490 µl HBSS- add 10 µl neutrophils then take 10 µl of diluted cells and add 10 µl trypan blue. Load 10 µl of cells/trypan blue onto a hemocytometer. Count the total number of cells in 4 large squares (edges comprising of triple lines and containing 16 smaller squares). Average that number and multiply by 106 = Nx106 cells/ml.