Development of graft-vs.-host disease-like syndrome in cyclosporine- treated rats after syngeneic bone marrow transplantation. I. Development of cytotoxic T lymphocytes with apparent polyclonal anti-Ia specificity, including autoreactivity

Cell Separation by Panning. Nylon wool-nonadherent spleen cells from the test animals were separated into lymphocyte subsets by panning, as previously described (20). Briefly, 10 7 lymphocytes, incubated (1 h at 4°C) with 0.2 ml of a 1:10 dilution (in phosphatebuffered saline [PBS]), were placed in petri dishes coated with affinity-purified goat antimouse IgG (Tago Inc., Burlingame CA), the plates were lightly centrifuged (200 g for 2 min) and incubated for 1 h at 4°C. The nonadherent fraction was aspirated, plates carefully washed with PBS, and the adherent fraction eluted by vigorous aspiration with a 10% solution of normal mouse serum or control ascites fluid. This fractionation procedure routinely achieved >90% purity of the selected cell population.