T-lymphocyte rosette inhibition titering of antisera by direct microassay

A 60-sample micro-rosette inhibition assay for determining the 100% rosette inhibitory titers of heterologous antisera is described. The assay is performed in histocompatibility trays, under oil, using frozen-thawed thymocytes or peripheral blood lymphocytes for an appropriate period, either with or without added complement; SRBC are inoculated into each well, and rosettes formed by centrifugation of the trays at 200 x g. following centrifugation, glutaraldehyde is added to each well to fix rosettes to the well bottom and the plates inverted to allow unbound SRBC to fall away. One hundred percent rosette inhibition is determined by low power microscopic examination of the inverted wells. Highly reproducible in-assay (+/-4% average standard error) and between-assay (+/-9%) inhibition values are obtained which correlated well (r = 0.99) with values calculated by more conventional methodology.