A fluorometric quantitative erythrophagocytosis assay using human THP-1 monocytic cells and PKH26-labelled red blood cells.

Removal of senescent, damaged or diseased red blood cells (RBCs) from the circulation in vivo occurs by a process known as erythrophagocytosis. The exact details of the signaling mechanisms that mark RBCs for recognition and influence erythrophagocytosis are still not completely understood. The aim of this study was to develop a quantitative, fluorometric erythrophagocytosis assay for human RBCs and phagocytes to aid elucidation of the biological mechanisms regulating erythrophagocytosis. RBCs were labelled with the lipophilic fluorescent dye PKH26 and incubated with the human monocytic cell line THP-1 at 37 degrees C for 45 min. Non-phagocytosed RBCs were lysed with hypotonic saline. Phagocytosed PKH26-labelled RBCs within THP-1 cells were detected with a fluorescence plate-reader and quantitated using a standard curve of known numbers of PKH26-labelled RBCs. Assay conditions were optimised for the numbers of phagocytes and RBCs, incubation time and fluorescence excitation and emission wavelengths. Erythrophagocytosis was also assessed by flow cytometry to determine the proportion of THP-1 cells with ingested RBCs and showed good correlation (P=0.7) between the two methods. The quantitative, fluorometric plate assay is very sensitive and has good reproducibility, making it a useful tool to investigate the biological mechanisms that regulate erythrophagocytosis of normal and diseased RBCs.