Bulk CFU culture and serial plating experiments were performed as in (37 (link)). In which, 100 HSCs (LSK+CD34−Flt3−CD150+CD48−) CD93+ or CD93− from 8- to 10-week-old mice were sorted by FACSAria Fusion II (BD). The cells were labeled or not with CFSE as indicated in the figure legends. Sorted HSCs were cultured in MethoCult (STEMCELL Technologies, #M3434) in technical duplicates. After 10 days in culture, the CFU capacity was evaluated by visual inspection. Then, we conducted serial plating with 10,000 cells originated from the primary CFU assay that were transferred to a new 24-well plate and cultured in MethoCult (STEMCELL Technologies, #03434). Next, after 10 days, colonies were quantified. In addition, downstream experiments to evaluate the daughter cells were conducted.
Protocol detail
Clonogenic Assay for Hematopoietic Stem Cells
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Corresponding Organization : Max Planck Institute of Immunobiology and Epigenetics
Other organizations : University of Freiburg
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Variable analysis
independent variables
- Presence or absence of CFSE labeling on sorted HSCs (LSK+CD34-Flt3-CD150+CD48-) CD93+ or CD93-
- CFU capacity of HSCs after 10 days in culture
- Colonies formed from 10,000 cells originated from the primary CFU assay after 10 days in culture
- Downstream experiments to evaluate the daughter cells
- Mouse age (8- to 10-week-old)
- Culture conditions (MethoCult STEMCELL Technologies #M3434)
- Technical duplicates for CFU assays
- Positive control: Not mentioned
- Negative control: Not mentioned
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