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

Area of Science:

• Immunology
• Hematology
• Cell Biology

Background:

• Antiserum against FMP1.1 mouse tumor cells cross-reacts with normal marrow progenitor cells (GM-CFC, BFUE).
• This antiserum also shows cytotoxicity towards colony-forming units (CFUs).

Purpose of the Study:

• To confirm immunological cross-reactivity of anti-FMP1.1 antiserum with CFUs using immunofluorescence.
• To optimize recovery of antibody-coated CFUs for analysis by fluorescence-activated cell sorting (FACS).
• To characterize the cell surface markers of CFUs and other hematopoietic stem cells.

Main Methods:

• Complement-dependent cytotoxicity assay.
• Immunofluorescent analysis using fluorescence-activated cell sorting (FACS).
• Enzyme digestion (papain) and antibody capping experiments.
• Dual parameter sorting (forward light scatter and fluorescence).

Main Results:

• Anti-FMP1.1 antiserum is confirmed to be cytotoxic to CFUs.
• Incubation with anti-rabbit IgG serum improved CFU detection by up to 67%.
• FACS enrichment yielded 26x for GM-CFC, 19x for BFUE, and 6x for CFUs.
• CFUs were identified in populations with high forward light scatter and low fluorescence.
• 5-fluorouracil (5-FU) treatment increased FMP1.1 immunofluorescence on bone marrow cells, notably on lymphocytes.

Conclusions:

• Anti-FMP1.1 antiserum identifies CFUs and other hematopoietic stem cells.
• Specific protocols enhance the detection and sorting of antibody-labeled CFUs.
• FACS analysis reveals distinct light scatter and fluorescence properties of CFUs.
• FMP1.1 expression is altered during bone marrow regeneration after 5-FU treatment.