Clonogenic methods in vitro for the enumeration of granulocyte-macrophage progenitor cells (CFU-GM) in human bone marrow and mouse bone marrow and spleen

Abstract

Protocols and techniques are described for the in vitro enumeration of granulocyte and macrophage progenitors found in human bone marrow and mouse bone marrow and spleen. Study of the colony forming unitgranulocyte-macrophage (CFU-GM) in human bone marrow is usually first accomplished by density separation of whole bone marrow “buffy coats” through Ficoll-Paque into low- and high-density fractions. After collection of the low-density fraction (containing most or all of the CFU-GM), cells are washed and suspended, at a known cell concentration, in a mixture of culture medium, fetal bovine serum (or serum-supplements), agar or agarose, with a source of colony stimulating factor(s). Cultures are allowed to solidify and are then placed in a humidified 37°C incubator at 5% CO2 in lowered (5%) O2 tension for 7 to 14 d. Colonies (>40 cells/ aggregate) and clusters (3 to 40 cells/ aggregate) are then scored. Murine CFU-GM are cultured and characterized in a similar manner except tissues are separated into a single cell suspension without a density separation, and the culture time is reduced to 5 to 7 d. Colonies and clusters are scored as previously described. Purification of CFU-GM can be performed and these cells cultured as above.