SIMULTANEOUS DETECTION OF CYTOKINE AND IMMUNOPHENOTYPE AT THE SINGLE-CELL LEVEL BY IMMUNOENZYMATIC DOUBLE STAINING

The goal of this study was to establish a generally applicable immunoe nzymatic method for the simultaneous detection of cytokine and immunop henotype at the single cell level. Evaluating various cell preparation s and staining protocols, we found that permeabilization by saponin (0 .1%) is very efficient, in combination with glutaraldehyde (0.04%) as fixative. Among various staining procedures, sequential immunoperoxida se labelling of the cytokine by use of diaminobenzidine, and detection of the immunophenotype by use of 4-chloronaphthol proved most discrim inative. The typical localization of the cytokine reaction product ('G olgi staining') within the cell, and the 'ringlike' staining for the i mmunophenotype on the cell surface, allowed precise identification of double-labelled cells. Primary monoclonal antibodies from the same spe cies could be used without loss of sensitivity and specificity for eit her or both antigens. This method thus provides the opportunity to stu dy morphology, cytokine and immunophenotype simultaneously at the sing le cell level with standard equipment. Its application for the analysi s of tissue samples is in progress, and may allow us to incorporate th e cytokine-type as a new parameter in histopathological diagnostics.