For cell cycle analyses, purified BM Lin− cells from WT and mutant mice were permeabilized and fixed according to the manufacturer’s instructions with the FOXP3 permeabilization kit (Foxp3/Transcription Factor Staining Buffer Set) and then labeled with Ki-67 antibody (clone B56, mouse IgG1; BD) and DAPI before flow cytometric analysis. For label-retaining assays (Wilson et al., 2008 (link)), mice were injected intraperitoneally with 180 µg BrdU (Sigma-Aldrich) and maintained on water containing 800 µg/ml BrdU and 1% glucose during 12 d. The BrdU pulse was followed by a 3-wk chase period to detect by flow cytometry LRC activity by combining surface staining to define ST-HSCs with intracellular staining using the BrdU-FITC labeling kit following the manufacturer’s instructions (BD). Cyclin D3 was detected by flow cytometry in fixed and permeabilized BM cells using a cyclin D3 mAb (clone 1/cyclin D3, mouse IgG2b; BD) followed by a secondary rat anti–mouse IgG2b PE-conjugated antibody (R&D Systems; Galloway et al., 2016 (link)). Apoptosis was measured using the active caspase-3–FITC apoptosis detection kit or the Annexin V apoptosis detection kit I (BD) followed by DAPI staining according to the manufacturer’s instructions as described elsewhere (Balabanian et al., 2012 (link); Biajoux et al., 2016 (link)).
Brdu fitc labeling kit
Manufactured by BD
The BrdU-FITC labeling kit is a laboratory tool used to detect and quantify cell proliferation. It contains reagents for the incorporation and detection of the thymidine analog bromodeoxyuridine (BrdU) into newly synthesized DNA of dividing cells. The FITC-conjugated anti-BrdU antibody allows for the fluorescent labeling and subsequent analysis of BrdU-positive cells.
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2 protocols using brdu fitc labeling kit
1
Analyzing Cell Cycle and Apoptosis in Hematopoietic Stem Cells
2
Comprehensive Cell Cycle Analysis
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For flow cytometry-based cell cycle analyses, bone cells were permeabilized, fixed with the FOXP3 permeabilization kit (Foxp3/Transcription Factor Staining Buffer Set; eBioscience), and labeled with a Ki67 Ab and DAPI. For BrdU assays, mice were injected intraperitoneally with 180 μg BrdU (Sigma) and maintained with drinking water containing 800 μg/ml BrdU and 1% glucose over 12 days. The BrdU labeling was analyzed by flow cytometry using the BrdU-FITC labeling kit (BD Biosciences). For in vitro BrdU incorporation, 3 μg/ml of BrdU was added to the culture and after five days the percentage of incorporation was determined as above. Apoptosis was measured using the Annexin V detection kit (BD Biosciences) with DAPI staining. For in vitro proliferation assays, SSCs were detached with 0.5% trypsin and loaded at 3 × 104 cells/well with cell trace violet (CTV, Thermofisher) for 15 min at 37 °C. CTV dilution was assessed by flow cytometry. To estimate the doubling time values, SSCs were seeded at 3 × 103 cells/cm2 and counted after 3 days of culture. The doubling time was calculated as follows: (time of culture × log(2))/(log(final number of SSC) − log(initial number of SSC)).
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