Equipment and reagents
• Various tissue culture vessels including 25 • RPMI1640 medium alone and containing and 75 cm2 flasks, 10 cm diameter Petri 15% heat-inactivated FBS
dishes, 24-well cluster plates • 2% gelatin tn double distilled H20
A Preparation of gelatin-coated plates
1 Add 2 g of type I gelatin to 100 ml of double distilled water and autoclave at 110°C for 40 min.
2 Bring the 2% gelatin solution to 50 °C.
3 Pipette 0.5 ml into 24-well tissue culture plates, 2 ml into 35 mm diameter plastic dishes. 10 ml to 75 cm2 tissue culture flasks, or 14 ml into 10 cm diameter Petri dishes.
4 Hold the gelatin-coated dishes at 50°C for 2 h.
5 Remove the excess gelatin by suction and incubate dishes at 374C, in a dry incubator, for 48 h before use. These pre-coated flasks can be stored under sterile conditions at room temperature for up to four weeks.
B Purification of macrophages
1 Rinse the gelatin-coated culture flasks twice with culture medium.
2 Add an appropriate volume of mononuclear cell suspension (see Protocol 1. footnote a) to each gelatin-coated vessel.
4 Remove the non-adherent cells by suction and gently wash flasks several times with culture medium pre-warmed at 37°C.
Production of microexudate is a property shared by a variety of mammalian cells of different origin. Accordingly, a number of human and murine cell lines can yield conditioned plastic surfaces suitable for purification of monocytes/ macrophages (19). The mechanism and specificity of attachment of these cells to microexudates is not completely understood. It is known that the composition of the microexudate is complex and contains FN, vitronectin, and possibly collagen amongst other molecules (50), Monocyte/macrophage adherence to these exudates is likely mediated by receptors for proteins of the integrin family. The presence of FN receptors may be important since neutrophils and lymphocytes bind poorly or not at all to FN-coated surfaces. Although serum proteins adsorb to plastic surfaces and have been considered to be a component of the microexudate, the different behaviour of monocytes/macrophages on coated versus uncoated surfaces in the presence of high serum concentrations indicates that the effect of the microexudate is not due simply to rapid adsorption of a serum component (19). Baby hamster kidney (BHK), mKSA-TUS, a Balb/c mouse kidney cell line, rat fibroblasts (R22CIF), and Chang liver cells are generally used as conditioning cell lines. Protocol 3 gives a procedure for the separation of macrophages on microexudate-coated surfaces.
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