Equipment and reagents
• 24-well cluster plates
• Collagen-coated plates
• RPMI 1640 medium alone and containing 15% heat-inactivated FBS
Protocol 4 continued
A Preparation of collagen-coated matrices
1 Dissolve lyophilized type I collagen at 1.5 mg/ml in 0.1 M HOAc at 4°C by stirring overnight.
2 Dispense 250 of type I collagen solution to each well of 24-well cluster plates.
3 Simultaneously, add 25 |jl1 of 10 x culture medium and 15 |xl of 0.142 M NaOH to each well to bring the pH of the mixture to 7.6,
4 Incubate plates for 1-2 h at 37°C (gelation usually occurs in 30-60 min), then wash well with culture medium. Collagen-coated dishes can be kept hydrated at 37°C until use.
B Purification of macrophages
1 Seed 3 X 106 Ficoll-Hypaque derived PBMC (see Protocol 8) per well in 1 ml of RPMI containing 15% FBS.
2 Incubate the plate at 37 °C for 1 h, to allow the attachment of monocytes.
3 Remove all non-adherent and loosely adherent cells by vigorous shaking the flasks and aspirate released cells.
4 Wash cell monolayer four or five times with medium without serum.
5 Check, under an inverted microscope, that non-adherent cells have been washed off and that most of the adherent cells are spread out.
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