Anti brdu fitc

For BrdU incorporation analysis, Grb10^m/+ mice or Grb10^+/+ mice were injected intraperitoneally with 2 mg BrdU suspended in PBS. At 24 hours, BM cells were collected and stained with anti-BrdU FITC (BD). For cell cycle analysis, BM cells were flushed into PBS plus 10% FBS and stained for surface c-kit, sca-1 and lineage markers for 30 minutes. Cells were then fixed with buffer III (BD) for 30 minutes, followed by incubation of permeabilization buffer (BD). Anti-Ki67 FITC (BD) was added to the cells and incubated for 30 minutes. 7AAD was added to stain for DNA content. For analysis of HSC apoptosis in Grb10^m/+ mice or Grb10^+/+ mice, BM cells were stained with anti-c-kit, anti-sca-1 and anti-lineage antibodies for 30 minutes, followed by application of the FITC Annexin V Apoptosis Detection Kit I (BD), as previously described (Quarmyne et al., 2015 (link)). For in vivo analyses, mice were irradiated with 700 cGy to allow sensitive detection of radiation effects on cell cycle or cell death in BM KSL cells. For in vitro cell cycle and apoptosis assays, BM c-kit⁺sca-1⁺lineage^- (KSL) cells were sorted into 96-well plate using a BD FACS Aria sorter. KSL cells were irradiated at 300cGy in vitro. At certain time points after irradiation, cells were collected and cell cycle and apoptosis assays were performed.

RPMI1640 medium containing 10% FCS was used for CD4 naïve T cell cultures. The following antibodies were purchased from BD BioSciences (San Jose, CA): anti-mouse CD3 (145-2C11), anti-mouse CD28 (37.51), anti-mouse IL-2, anti-CD4-PerCP, anti-CD4-PECy7, anti-CD8-FITC, anti-CD25-APC, anti-CD25-PE, anti-CD44-FITC, anti-CD62L-PE, anti-BrdU-FITC. Anti-FoxP3-APC, anti-IFNγ-APC, and anti-IL-17A-PE were from eBioscience (San Diego, CA). Anti-Helios, anti-CD122 and anti-GITR were from Biolegend (San Diego, CA). Recombinant mouse IL-2 and IL-7 were purchased from R&D Systems (Minneapolis, MN) and ovalbumin was from Sigma-Aldrich (St Louis, MO).