Enrichment of human intestinal mast cells can be performed by using magnetic cell sorting (MACS). In general, mast cells are 50-75% pure after positive sorting by immunomagnetic labeling of c-kit (15,16). In some cell preparations, the purity can be greater than 90%. Further purification, as much as 100%, is possible by culture of the cells (see Subheading 3.3.).
Although this method is a powerful tool for the enrichment of human intestinal mast cells it has also some limitations: (1) recovery of the mast cells is low (30-50%); (2) the positive fraction containing the mast cells frequently are contaminated with high amounts of dead cells and cell debris that
nonspecifically bind to the magnetic beads; and (3) the functional response to physiological activators and the culture sufficiency is diminished directly after MACS purification but can be restored after culture (15,16).
Purification of mast cells can be achieved also by long-term culture of nonenriched cell fractions (19). This approach has the advantage of higher cell numbers and a better culture sufficiency. The disadvantages are that the purity is often poor and the required culture period quite long. This issue will be discussed in detail in the following section.
MACS is the method of choice for the purification of mast cells if long-term culture needs to be avoided.
1. Culture freshly isolated intestinal cells at 4 X 106/mL in culture medium overnight in 75-cm2 (up to 1.2 X 108 cells) or 150-cm2 (up to 2.5 X 108 cells) tissue culture flasks (see Note 5).
2. Harvest cells by gently scraping with a plastic cell scraper (see Note 6). Count after staining with trypan blue (see Note 7).
3. To remove clumps, pass cells through a 30-pm filter. Centrifuge cells (300g, 10 min, 4°C) and remove supernatant. Keep cells cold during the following steps (use cold solutions, centrifugation at 4°C, and so on).
4. Resuspend cells in MACS buffer (200 pL/108 cells). For fewer cells, use 200 (L. Add MAb YB5.B8 (1.25 pg/108 cells), mix well, and incubate for 15-20 min in a refrigerator at 4-10°C.
5. Wash cells with 10-20 mL of MACS buffer, remove supernatant, and resuspend in MACS buffer (200 (L/108 cells) again. For fewer cells, use 200 pL. Add MAb Goat Anti-Mouse IgG MicroBeads (1.25 (g/108 cells), mix well, and incubate for 15 min at 4-10°C (see Note 8).
6. Wash cells with 10-20 mL of MACS buffer, remove supernatant, and resuspend in 5 mL of MACS buffer/108 cells. For fewer cells, use 5 mL.
7. Place a positive selection column type LS+ in the magnetic field of an appropriate MACS separator (located in a 4°C room). Wash with 5 mL of HA buffer according to the manufacturer's instructions. Discard eluate.
8. Transfer as many as 108 cells to the top of the MACS column (see Note 9). Once the cell suspension has completely entered start washing of the column with at least 10 mL MACS buffer. Collect the effluent in a 50-mL Falcon tube (mast cell-depleted fraction).
9. Remove MACS column from the separator. Fill column with MACS buffer (as much as 7 mL), firmly flush out the positive fraction using the supplied plunger and collect cells in an appropriate tube.
10. Centrifuge cells and resuspend in culture medium. Count the cells after staining with trypan blue. Prepare cytocentrifuge smears and stain with May-Grunwald/ Giemsa to perform a differential count (see Note 10).
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