Comparison of two fluorochromes for flow cytometric assay of cellular glutathione content in human malignant melanoma.

Because the resistance of melanoma to cytotoxic therapy may be due to high intracellular glutathione (GSH), we wished to monitor GSH in melanoma cells during treatment. This required an assay suitable for very small samples. So we chose analytical flow cytometry. We calibrated the flow cytometric assays against biochemically determined GSH content in a range of cultured human melanoma cell lines, then applied the assays to fine-needle tumour biopsies. Mercury orange was the first fluorochrome suitable for use in a flow cytometer with a 488 nm light source, but many technical factors influenced the results. Chloromethyl fluorescein diacetate (CMFDA) allowed assay conditions less dependent on details of cell handling. Correlations between biochemical GSH content and fluorescence intensities in cell lines were good for mercury orange and CMFDA. CMFDA is the preferred fluorochrome for estimating cellular GSH content in biopsies from human melanomas. Tumours metastatic to or beyond regional lymph nodes had significantly more cells with high GSH than primary tumours or regional recurrences.