The following protocol is used to purify white blood cells from buffy coat. It will render monocytes, lymphocytes and a few eosinophils.
If further purification of monocytes is needed, a method based on attachment to surfaces is explained later.
- 50-ml polypropylene tubes
- Sterile disposable 5- and 10-ml plastic pipettes
- Sterile 21G needle and 50-ml syringe
- Adjustable 20-µl, 200-µl and 1000-µl pipettes and sterile disposable tips
- Sterile disposable 1.5- and 5-ml tubes
- 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)
- May-Grunwald stain
- Giemsa stain
1. Draw buffy coat of blood derived from a donor contained in a bag with a 50-ml syringe.
2. Place up to 20 ml of buffy coat in a 50-ml tube.
3. Add approximately 6 ml HBSS- 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!
4. Centrifuge at 1,300 rpm for 30 minutes. After centrifugation the plasma, the buffy layer and Ficoll should have formed distinct layers in that order from the top downwards and the neutrophils along with the remaining erythrocytes will be present in a pellet at the bottom.
5. Aspirate off the top layer of plasma taking care not to aspirate the buffy layer. The thickness of the buffy layer will depend on the cell count of the donor from whom the blood was collected.
6. Collect buffy layer using a sterile disposable pipette. This is best achieved by aspirating the monocytes from the bottom of the layer whilst turning the tube, do not aspirate all of the Ficoll layer.
7. Transfer monocytes to a fresh 50-ml sterile disposable tube and add up to 40 ml HBSS-.
8. Pellet the monocytes by centrifuging at 1,300 rpm and 4oC for 10 minutes. Aspirate the supernatant.
9. Resuspend in HBSS- (1-3 ml depending upon the size of the pellet), store on ice and use quickly.
10. Quantify the total cell number per ml as follows:
To 490 µl HBSS- add 10 µl buffy layer, 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.
11. Estimate the percentage of monocytes in the buffy layer as follows:
Smear 10 µl of the cell suspension onto a glass slide and dry. Immerse in May-Grunwald stain for 10 minutes followed by Giemsa stain for 30 minutes. Rinse in water and dry. Microscopically, monocytes have an irregular cell margin with a non-lobular but non-spherical nucleus as compared to B or T cells and large granular lymphocytes (LGL) which have a spherical shape with a large circular nucleus; by comparison neutrophils and eosinophils have a spherical shape and a highly lobular nucleus. A total of 100 cells are counted divided into the number of monocytes and other lymphocytes, from which monocytes can be expressed as a percentage of the total cells/ml.
Further purification of monocytes based on attachment
When performed on the white blood cells purified as outlined above, it will render approximately 95% monocytes.
- White blood cells
- Tissue culture flask or tissue culture dish
- Warm HBSS-
- Warm fetal calf medium (FCM; RPMI 1640 supplemented with 10 % Fetal
- Calf Serum, 100 U/ml penicillin and 100 µg/ml streptomycin)
- Disposable cell scraper (i.e. Sarstedt, Costar, Corning Inc., NY., U.S.A.)
1. Place the white blood cells in a flask or culture dish at a maximum concentration of 2x106 monocytes per ml in FCM.
2. Incubate at 37oC with 5% CO2 for 2 hours.
3. Shake the flask gently and aspirate the medium. Add warm HBSS-, shake gently and aspirate. Wash once more.
4. Add 10-20 ml of cold HBSS- and gently scrape all the surface of the flask.
5. Transfer the liquid to a 50-ml tube.
6. Add 10 ml of HBSS- to the flask, shake it gently and again transfer the liquid to the 50-ml tube.
7. Centrifuge for 10 minutes at 1,500 rpm. Aspirate the supernatant and resuspend the pellet.
8. Count cells as outlined above.