WT bone marrow cells (BMCs) were seeded on top of confluent WT, Fanca−/− and Fancc−/− MSCs in 35 mm culture dish (BD Falcon, Tewksbury, MA) and allow the precursor cells forming hematopoietic clones under the stromal layers [19 (link), 32 (link)]. The cells were cocultured in 37°C incubator supplemented with 5% CO2 and fed weekly by half medium change. Phase-dark hematopoietic clone was imaged under phase contrast images were taken at 20× objective. The identified area was analyzed with ImageJ software (NIH).
Protocol detail
Coculture of BMCs on Fanconi Anemia MSCs
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Corresponding Organization : West Virginia University
Other organizations : South China Normal University
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Variable analysis
independent variables
- Genotype of the stromal cells (WT, Fanca-/-, Fancc-/-)
- Formation of hematopoietic clones under the stromal layers
- Bone marrow cells (BMCs) used were from WT mice
- Cells were cocultured in a 37°C incubator supplemented with 5% CO2
- Cells were fed weekly by half medium change
- Positive control: WT stromal cells
- Negative controls: Fanca-/- and Fancc-/- stromal cells
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