Use of lucifer yellow iodoacetamide in a flow cytometric assay to measure cell surface free thiol.

Standard measurements of free thiol on cell surfaces use the uptake of thiol-reactive substances which are incapable of traversing the membrane (reviewed in [l]). One problem is that dead cells are incapable of excluding such reagents, so that there is a risk of systematically high bias in the results, especially in delicate cells from patients. For example, one group showed a 3OOO% increase in uptake when the concentration of reagent was increased the to a toxic level[2]. It should be possible to overcome this problem by using a fluorescent thiol reagent in a flow cytometric assay. Appropriate gating allows estimates of available thiol in live and dead cells, and also comparison of surface thiol in different cell types in a single preparation. These experiments test the value of one such reagent, lucifer yellow iodoacetamide (LYIA). We measured the effects of large amounts of external thiol on LYIA uptake, and also the effects of plasma thiol concentration on leucocyte surface thiols in situations where the concentration is expected to be low. LYIA proved toxic to cells at concentrations above 150 pM and it was not found possible to devise a system in which 'total' available surface thiol could be shown to react. In a standard experiment, cells were washed in HBSS, pH 6.8, resuspended at 1.5 x 107/ml, incubated with LYIA (100 pM, pH 7.6) for 30 minutes, washed twice in Hanks buffered salt solution (pH 6.8), transferred to a fresh tube, washed once more and analysed in an EPICS-C flow cytometer (EX 455nm, EM 540 nm with power set to 200 MW at 100 A). For cultured cells, gating was set to measure live and dead cells separately, while for peripheral blood lymphocytes, it was set to measure 3 populations which contain predominantly either lymphocytes, monocytes or neutrophils. In a comparison of 5 cultured cell lines (Table 1) uptake by live cells correlated with size. Uptake by dead cells was usually higher, particularly by an endothelial cell line.