By R. Doyle, I. Ofek
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The final supernatants are collected into 10 × 75 mm glass tubes and saved on ice. 6. Five hundred microliters of this material is mixed with 50/zl perchlotic acid and vigorously vortexed. 7. This sample is centrifuged at 800 g for 10 min, and 300/xl of the supernatant is transferred to a new tube. (At this point, samples can be stored at - 2 0 ° or -70°). 8. One hundred microliters sodium hydroxide and 400/xl O P T are added to the sample or to 300/~1 of known amounts of histamine (standard), and the resulting mixture is vortexed and kept on ice for 40 min.
A. Panley, Infect. Immun. 25, 738 J0 I. E. Salit and E. C. Gotschlich, J. Exp. Med. 146, 1169 (1977). 11 T. K. Korhonen, V. Vaisanen, H. Saxen, H. Hultberg, and S. B. Svenson, Infect. 37, 286 (1982). 12 j. p. Duguid, S. Clegg, and M. I. Wilson, J. Med. Microbiol. 12, 213 (1979). 13 I. Orskov, A. Birch-Andersen, J. P. Duguid, J. Stenderup, and F. Orskov, Infect. 47, 191 (1985). Immun. (1979). Immun. Immun. 46 G E N E R A L METHODS FOR A D H E S I O N TO A N I M A L CELLS  Estimation of Binding of Purified Adhesins to Red Blood Cells or to Erythrocyte Membranes Binding of Red Blood Cells to Purified Adhesins (Hemadhesion).
The abdomen is gently massaged and the peritoneal lavage from each mouse is collected and pooled in a 50-ml tube. 4. The pool of peritoneal cells is centrifuged at 450 g for 10 rain at 25° and suspended in 2 ml TG buffer. 5. The suspension is gently layered over 2 ml of the metrizamide solution in a 15-ml tube. 6. The tube is centrifuged at 450 g for 20 min (25°). 7. The peritoneal cells, which collect at the interface, are aspirated carefully and discarded. The supernatant is also aspirated, and a cotton swab is employed to remove all residual cells attached to the walls of the tube.