Tumor-associated surface membrane antigens on CFUs: characterization using the fluorescent activated cell sorter

Antiserum raised in rabbits against the FMP1.1 mouse tumor cell line is known to cross-react with GM-CFC and BFUE from normal marrow using the complement-dependent cytoxicity assay. It has been found that this antiserum is cytotoxic to CFUs, either with or without complement addition in vitro. Immunofluorescent analysis using the fluorescent activated cell sorter (FACS) has confirmed this immunological cross-reactivity with CFUs. Recovery of antibody-coated CFUs posed a potential problem in FACS experiments due to in vivo destruction by either opsinization or cytotoxic lysis. Experiments involving enzyme digestion (papain) of antibody and preincubation of bone marrow cells to allow antibody capping did not show improved recovery of CFUs. However, incubation of anti-FMP1.1 coated bone marrow cells with specific anti-rabbit IgG serum resulted in up to 67% of CFUs being detected. Dual parameter sorting with FACS, involving forward light scatter and fluorescence, gave about 26 and 19 times enrichment of GM-CFC and BFUE, respectively, whereas CFUs were enriched by only 6 times. These colony-forming cells were found in the cell population with high intensity forward light scatter and with relatively low fluorescence. Bone marrow cells undergoing regeneration 7 days after 5-fluorouracil (5-FU) treatment of mice exhibited more intense immunofluorescence than normal marrow cells. In particular, a distinct population of lymphocytes in 5-FU treated marrow reacted with anti-FMP1.1 serum, which was not observed with normal marrow.