FLOW CYTOMETRIC DETECTION OF 2 TYPES OF TUMOR-NECROSIS-FACTOR-ALPHA (TNF) RECEPTORS BY BIOTINAMIDOCAPROYL-TNF IN HUMAN TUMOR-CELLS

Recombinant human tumor necrosis factor-alpha (TNF) was reacted with N HS-biotin and NHS-biotin-amidocaproate to yield the derivatives of TNF which in conjunction with avidin-FITC and flow cytometry were tested for labeling of the cell preparations bearing TNF receptors (TNFR). Th e human epithelial (HEp-2, HeLa, MCF-7) and myeloid (HL-60, U937, K562 ) cell lines bearing TNFR55 and TNFR55/75 respectively, were selected for this purpose. As judged from competitive experiments, both biotiny lated forms of TNF specifically bind to receptors, however only the bi otinamidocaproyl-TNF was recognized by avidin-FITC on the surface of a ll tested cell lines, whereas biotinyl-TNF stained myeloid cells, but not cells of epithelial origin. Based on the reported data about diffe rent sensitivity of two types of TNFR to trypsin digestion, the effect of trypsin treatment on the following TNFR labeling was examined. Aft er preliminary optimized mild trypsinisation, TNFR staining was comple tely abrogated in the TNFR55-bearing epithelial cells, but only partia lly decreased in the TNFR55/75-bearing myeloid cells. On the grounds o f these observations, we developed a simple approach for the estimatio n of relative expression of the two types of TNFR on the cells employi ng the only biotinylated TNF as specific probe.