Flow cytometry with crystal violet to detect intracytoplasmic fluorescence in viable human lymphocytes demonstration of antibody entering living cells

Abstract

A new method is described for the detection of intracytoplasmic flourescence and its differentiation from surface staininng of viable human lymphocytes using flow cytometry after addition of crystal violet which quenches surface but not internal fluorescence. This has then been used to study antibody penetration of viable human lymphocytes, using FITC-conjugated IgG from normal serum or serum containing anti-RNP antibody. The results showed that 54 ± 1% normal lymphocytes were penetrated by anti-RNP antibody and 23 ± 3% by normal IgG respectively. The lymphocyte population analysed by flow cytometry has been directly demonstrated to be viable by FDA staining. These results provide unequivocal evidence that antibody can penetrate viable human lymphocytes.