Perm wash buffer 1

Mice were exposed to one dose of 7 Gy radiation and bone marrow was harvested 24 hours later. Cell cycle analysis of HSC, HPC, MPPs and LK compartment were assessed by Pyronin Y (0.25µg/mL/10⁶ cells) (Sigma-Aldrich, St. Louis MO, USA) and Hoescht33342 (2µg/mL/10⁶ cells) (Invitrogen) dyes. For intracellular analysis of the phosphorylated state of p38 protein into HSCPs, surface antigen-labeled cells were fixed with Cytofix buffer (BD Biosciences) for 20 min and then permeabilized using Cytofix/Cytoperm buffer (BD Bioscience) for 20 minutes. After washing, cells were stained intracellularly using Alexa Fluor® -647 anti-phospho-p38(clone 36/p38 (pT180/pY182), BD Biosciences) and Alexa Fluor® -647 anti-phospho-ERK1/2 (clone 20A, BD Biosciences) for 40 minutes in Perm/Wash Buffer 1× (BD Bioscience) with 0.5% of mouse serum. All incubations after cell stimulation were done on ice and in the dark. Cell acquisition was performed by flow cytometry (LSRFortessa I, BD Biosciences) equipped with FACSDIVA™ software (BD, Biosciences) for multiparameter analysis of the data.
For in vitro quantification of apoptotic mouse embryonic fibroblast cells, an Alexa Fluor-647 labeled cleaved caspase 3 antibody (Cell Signaling #9602, Danvers, MA, USA) was incubated on methanol-permeabilized cells for one hour and analyzed on a BD-FACSCanto II instrument, as reported previously.^{[17 (link)]}

Staining with 7-AAD and anti-Ki67 antibody (SolA15) was performed after fixation/permeabilization with the Foxp3 staining buffer set (eBioscience). Staining for phosphorylated BCR signaling components was performed on peritoneal cells isolated with ice-cold FACS buffer (2% FCS in PBS) and stained for CD19, B220 and CD5 on ice. The ice-cold cell suspension was mixed with an equal volume of pre-warmed Cytofix buffer (BD) and fixed for 15 minutes at 37 °C. Cells were washed with Perm/Wash Buffer I (BD) and stained in this buffer with antibodies against IgM and phosphorylated BCR signaling components for at least one hour at 23 °C.

Decidual leukocytes were thawed, then washed with FACS buffer and surface stained for CD14 and HLA-DR in FACS tubes. Pellets were washed in FACS buffer and resuspended in 100 μL prewarmed PBS (Ca+ Mg+ free). Cells were fixed immediately by the addition of equal volumes of prewarmed Cytofix Buffer (BD Biosciences) and thorough mixing and incubating at 37C for 10 min. Cells were then centrifuged at 600g for 8 min. Supernatants were removed leaving no more than 50 μL residual volume. Cells were then permeabilized by the addition of 1 mL 1X BD PermWash Buffer I, mixed well, and incubated at RT for 30 min. Pellets were then spun, aspirated, and stained intranuclearly with antibodies against NF-κB p65 (pS529) AF647 (Clone K10–895.12.50, Cell Signaling Technology) or IκBa PE (Clone MFRDTRK, eBioscience, San Diego, CA) and Phospho-p38 MAPK-APC (Clone 4NIT4KK, eBioscience) for 1h at RT in the dark. Samples were washed twice in PermWash Buffer I, resuspended in FACS buffer and acquired on the Attune NxT flow cytometer.

Monocytes were identified using HLA-DR, CD14 and CD16, as described above. Cell surface expression of the IFNγ receptor R1 was determined with IFNγR1 antibody (CD119, R&D Systems, Minneapolis, MN). For intracellular detection of SOCS3, cells were permeabilized with Cytofix/Cytoperm Kit (BD Biosciences), stained for SOCS3 (Abcam, Cambridge, MA), followed by APC-labeled goat anti-rabbit antibody (R&D Systems). For intracellular detection of total STAT1, BD PhosphoFlow Fix Buffer I and Perm/Wash Buffer I were used. Cells were stained for STAT1 (Cell Signaling Technologies, Beverly, MA), followed by FITC-labeled goat anti-mouse antibody (Invitrogen, Carlsbad, CA).