Flow check fluorospheres

Flow cytometry was used to confirm neutrophil depletion following anti–Gr-1 and anti–Ly-6G treatment. To prevent clotting, 20 μl whole blood was mixed with an equal volume of sodium citrate and kept on ice. Samples were incubated with PE-labeled rat anti-mouse CD45 Ab (1:100; BioLegend) and Pacific Blue–labeled rat anti-mouse Ly-6G Ab (1:100; BioLegend) for 30 min on ice. RBCs were then lysed, and the cells were fixed by the addition of 1 ml BD FACSlyse solution (BD Biosciences, Oxford, U.K.). Samples were centrifuged for 5 min at 350 × g, the supernatant removed, and the samples resuspended in 200 μl PBS for flow cytometric analysis. Prior to analysis, 50 μl Flow-Check fluorospheres (Beckman Coulter, High Wycombe, U.K.) was added to each sample to allow subsequent calculation of the absolute number of cells counted. Analysis was carried out using the BD LSR Fortessa (BD Biosciences), and data were collected using BD FACSDiva software (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). Granulocytes, monocytes, and lymphocytes were gated based on their forward and side scatter. Neutrophils were specifically gated based on expression of both CD45 and Ly-6G.

After collagenase digestion of the right lung, red blood cells were lysed (ACK lysis buffer, Invitrogen) and Fc-mediated antibody binding reduced by incubation with Fc block (Mouse BD Fc block, BD Bioscience). Lung digest cells, BAL fluid cells and whole blood were incubated with anti-CD45 and anti-Ly6G antibodies (Biolegend, London, UK) for 30 min at 4 °C in the dark. After addition of FACSlyse (BD Bioscience) cells were resuspended in PBS and 50 μl flow-check fluorospheres added (Beckman Coulter, Brea, CA) prior to flow cytometric analysis.

CEM cells either pre-incubated with DMSO or 5 μM Y27632 (Calbiochem) overnight or transfected with different plasmids were deposited in the upper compartment of a Transwell insert (pore diameter: 5 μm; Nunc). The lower compartment contained no chemokine or 200 ng/ml CXCL12. After 3 h of incubation, the inserts were removed, an equal amount of calibration beads (Flow-Check Fluorospheres, Beckman Coulter) was deposited in the wells, and the number of cells having migrated to the lower compartment relative to the number of beads was quantified by flow cytometry (FACS Calibur, BD).
For Y27632 experiments, migration was quantified as the number of migrating cells/total cell number. For each transfectant, an eventual bias in cell migration due to Fam65b expression was quantified by calculating the following formula: migration index = % of migrating GFP⁺ cells/% of migrating GFP⁻ cells.

After single GO-ATeam2 knock-in HSC culture, most of the medium (150–170 µL) in wells of a 96-well plate was aspirated and samples were stained with 10 µL antibody cocktail for 30 min at 4 °C. Antibodies used were anti-lineage markers (CD4, CD8a, Gr-1, Mac-1, B220, Ter-119)-PerCP-Cy5.5, anti-c-Kit-APC-Cy7, anti-Sca-1-PE-Cy7, anti-CD150-BV421, and anti-CD48-APC for LSK-SLAM analysis. Antibody cocktail was prepared by mixing 0.1 µL of each antibody. After incubation, 100 µL PBS +2% FCS was added to wells, and the plates were centrifuged for 5 min at 4 °C and 400 × g with low acceleration and medium deceleration. Then, 100 µL supernatant was aspirated and cell pellets were resuspended in 200 µL PBS +2% FCS+0.1% PI+0.25% Flow-Check Fluorospheres (Beckman Coulter, Brea, CA, USA, Cat# A69183). Samples were acquired in fast mode in the MACSquant analysis settings, and volumes of 100 µL (large colonies) or 150–170 µL (small colonies) were analyzed. Data were exported as FCS files and analyzed using FlowJo software. Cell number was corrected by bead count of Flow-Check (~1000 cells/µL). HSCs were counted using CD150⁺CD48^-LSK cell counts. Megakaryocytes were identified as cells with high forward scatter and side scatter, as well as high CD150 and CD41 expression.