Two-step leukocyte migration in agarose technique.

A two-step modification of Clausen's technique of leukocyte migration under agarose is described. Blood leukocytes from a subject are incubated for approximately 4 days with or without antigen. The cultures, after reconstitution with antigen followed by repeated washing of the cultured cells, are added to fresh, dextran-separated blood leukocytes, and migration tests are performed in the absence of the antigen. Cells preincubated with antigen give a very marked reduction of the migration of the target leukocytes which must not necessarily be autologous. An inhibition of target cell migration can be seen with cells cultured without antigen for up to 3 days; with longer culture time, this effect is reduced or disappears. The effect of cells cultured with antigen, however, is equally strong irrespective of the culture time up to 1 week, and the most clearcut results will, therefore, be obtained after approximately 4 days culture. Also migration enhancement has been seen. Treatment of cells during the incubation with antigen and cycloheximide gives a dose-dependent blocking of the inhibiting effect. 5 patients were studied both before and after sensitization with DNCB: low concentrations of DNCB gave an inhibition and high concentrations an enhancement of the migration when the patients were sensitized. The technique is more sensitive and reproducible than the original Clausen technique. It is also suitable in studies where shortage of cells is a problem.