MOLECULAR MECHANISMS OF ERYTHROPHAGOCYTOSIS - FLOW CYTOMETRIC QUANTITATION OF IN-VITRO ERYTHROCYTE PHAGOCYTOSIS BY MACROPHAGES

A rapid, sensitive, and reproducible flow cytofluorimetric procedure i s described for quantitation of erythrophagocytosis based on the use o f red blood cells (RBCs) labeled with the fluorescent probe PKH-26. Th e procedure involves the following steps: i) incubation of PKH-26-labe led erythrocytes with macrophages, ii) removal of un-bound red blood c ells, iii) lysis of membrane-bound RBCs, and iv) measurement of extent of phagocytosis by direct flow-cytometric analysis of intact macropha ges. Each step was controlled by fluorescence microscopy, Use of fluor escent, instead of radio-labeled RBCs, makes the method more sensitive , rapid, and avoids radioactive hazards, Furthermore, this approach is multi-parametric and can distinguish different populations of macroph ages with reference to their erythrophagocytic potential, This technol ogy moreover, has broad applications from the initial step of contact (between effector and target cells to study the specific receptor medi ated attachment) to the subsequent cascade of time-dependent changes r esulting from that signal transduction, (C) 1997 Wiley-Liss, Inc.