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

Scott Cooper, Hal Broxmeyer

Research output: Contribution to journalArticle

18 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)77-81
Number of pages5
JournalJournal of Tissue Culture Methods
Volume13
Issue number2
DOIs
StatePublished - Jun 1991

Fingerprint

Granulocyte-Macrophage Progenitor Cells
Macrophages
Bone
Spleen
Bone Marrow
Colony-Stimulating Factors
Incubators
Ficoll
Serum
Sepharose
Agar
Purification
Culture Media
Cultured Cells
Suspensions
In Vitro Techniques
Tissue

Keywords

  • bone marrow
  • cultures
  • hematopoietic progenitors
  • spleen

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Anatomy

Cite this

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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.",
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author = "Scott Cooper and Hal Broxmeyer",
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AB - 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.

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