* 13 mm, no.l thickness glass coverslips • 24-we 11 dishes (Falcon, Becton Dickinson)
A Preparation of coverslips
1 The coverslips should be sterilized and cleaned before use by washing in ethanol and flaming,
2 To prevent macrophages attaching to the undersurface of the coverslip, place coverslips in the wells of a 24-well plate, cover with culture medium, and press down onto the well bottom before adding the cells.3
1 Staining of a coverslip can be done either in a well of a 24-well dish or by floating the coverslip on a drop of antibody pipetted onto Parafilm. It is quicker and easier to stain the coverslip in the well and it does require less handling of the fragile coverslip, though larger volumes of antibody for staining are needed. Staining on a drop of Ab cannot be used when Triton X-100 has permeabilized the cells as the presence of detergent reduces the surface tension, making it difficult to float the coverslips.
2 The coverslips can be mounted on glass slides for easy handling; plastic coverslips with suitable optical properties can also be mounted on glass. If analysing the cclls by flow cytometry do not plate the macrophages on coverslips, but on a substratum from which the cells can be detached (see Section 2.3.2).
3 The suggested plating density for each well is 2-5 X 10s macrophages.
By their nature, macrophages are uniquely adherent cells and are difficult to detach from plastic dishes. Cultivation in scrum makes it easier to detach macrophages compared with serum-free protein-containing medium such as Optimem-I (Gibco). Dishes may be coated with proteins such as fibronectin, microexudate, or gelatin (plus serum as a source of adhesion molecules; see Chapter 2). It is often beneficial to culture the macrophages on bacteriological, i.e. unmodified plastic surfaces. The complement receptor type 3 (CR3) is responsible for most of the adhesion to serum-coated bacteriological plastic, while the macrophage scavenger recoptor (SR-A) mediates most macrophage EDTA-rcsistant adhesion to serum-coated tissue culture plastic dishes. Our laboratory uses a combination of EDTA and Lidocaine-HCl, to avoid the use of degradative enzymes, like trypsin, that may destroy antigen epitopes and are ineffective (6). Lidocaine-HCl can also be added directly to serum-containing culture medium. Scraping cells from the bottom of the dish may damage fragile and more spread cells in the population; rather pipette the Lidocaine/EDTA vigorously over the cells. Procedures can also be performed at 4°C, with pre-cooled cells. The viability of the cells should be tested in all cases. Pro toco! 2 describes the use of Lidocaine for detaching macrophages.
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