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Deplli from the meniscus (mm) aller centrifugitig

Deplli from the meniscus (mm) aller centrifugitig

Fig. 5. Separation of human blood components by centrifuging on a Percoll® density gradient. Blood was overlaid on a preformed 70% (v/v) Percoll® density gradient in 0.15 mol/L NaCl and centrifuged at 400g for 5 min, and then the plasma layer containing platelets was removed and centrifugation was continued at 800g for 15 min, resulting in isopycnic banding of the blood cells. Redrawn from (47).

recovered by positive immunoselection, or irrelevant cells can be removed by negative selection using a cocktail of lineage-specific antibodies in a number of different ways (e.g., by immunoagglutination, with antibody-coated magnetic beads, or by immunoaffinity chromatography) (Fig. 6; ref. 49). When it is necessary to enrich samples in this way prior to cell-sorting (as it frequently is) or when preparations of 90-95% purity will suffice for experimental purposes, a commercially available automated immunomagnetic "pre-sorter" (autoMACS Pre-Sorter; Miltenyi Biotec Inc., Auburn, CA) can be used. Examples of some of the reagents and kits that are available commercially for this purpose are given in Table 2.

3.1.2.7. Erythrocyte Lysis

Erythrocytes in whole blood can be lysed with water, isotonic ammonium chloride, and acid (e.g., formic acid) or by permeabilizing their membranes with saponin; however, 100% lysis is rarely achieved and a small number of erythrocyte "ghosts" usually contaminate the remaining leukocyte preparation.

Fig. 6. Negative-selection principle using antibody-coated, superparamagnetic beads. The sample is labeled with an antibody specific for lineage antigen present on the unwanted cells which has been coupled to magnetic colloid beads (A, B). Placing the tube containing the cell suspension in a strong magnet for a few minutes causes the unwanted cells to be held on the sides of the tube, allowing the unlabeled cells of interest to be decanted (C).

Fig. 6. Negative-selection principle using antibody-coated, superparamagnetic beads. The sample is labeled with an antibody specific for lineage antigen present on the unwanted cells which has been coupled to magnetic colloid beads (A, B). Placing the tube containing the cell suspension in a strong magnet for a few minutes causes the unwanted cells to be held on the sides of the tube, allowing the unlabeled cells of interest to be decanted (C).

The expression of many antigens is unaffected by these procedures, but some reagents can affect some leukocyte functions (e.g., oxidative burst and degranulation) and can affect the apparent expression of some platelet and neutrophil antigens (50). When considering whether to use these techniques, it is prudent to check their effects using live leukocytes that have been prepared by another procedure. Some commercially available erythrocyte lysing reagents are listed in Table 3.

Table 2

Some Commercially Available Reagents for the Selection of Leukocyte Populations

Product

Supplier

Comments/intended use

BioMag®

Dynabeads®

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