Facs gallios system

Bone marrow cells were flushed with PBS/2% FCS from femurs and tibias and treated as above. Spleen tissue was minced and squeezed through a 100 µm cell strainer to obtain single cell suspensions. After lysis of erythrocytes with 3 successive incubations (2 min, RT) in AKC-lysis buffer (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7,3), cells were washed with PBS/2% FCS and live cells were counted with a CASY Cell Counter (Omni Life Science). Per 1 × 10⁶ cells 0.5 µl of the respective antibody was used, except for CD48, CD117, CD16/32, CD150 and Sca-1 (1 µl per 1 × 10⁶ cells) or CD34 (2 µl per 1 × 10⁶ cells). Incubation time was 15 min at RT, except for CD34 (1.5 h at 4 °C). The biotin-labelled Sca-1 antibody was stained with Streptavidin-PE-Cy7 for 15 min. For progenitor cell analysis, CD34 staining was done first followed by the addition of further antibodies. The gating strategy is outlined in Fig. 2a. Flow cytometry measurements were conducted with a FACS Gallios system (Beckman Coulter) and analyzed with FlowJo or Kaluza Software.

Cells (1 × 10⁶/ml) were either stimulated with 2.5 ng/ml PMA and 1 μg/ml ionomycin or left unstimulated. Cells were collected (5 × 10⁵ cells per sample), washed twice with PBS, and fixed with 0.4 ml 3.7% paraformaldehyde for 20 min at room temperature. After two additional washes, cells were permeabilized with 0.1% Triton X-100 in PBS for 4 min at room temperature. Cells were blocked for 45 min with 2% goat serum, followed by 45 min incubation with 0.1 μg mouse anti-WASp Ab, on ice. The samples were washed twice, and then incubated with 1:2000-diluted Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (Jackson), for 45 min on ice. Fluorescence was analyzed by flow cytometry on a FACS Gallios system (Beckman Coulter).

For the assessment of apoptotic cells, isolated BM cells were applied to MACS lineage depletion as for cell sorting (see above) and 3 × 10⁵ lin⁻ cells were seeded in 300 µl StemSpan^TM medium supplemented with each 20 ng/ml IL-3 and IL-6 and 50 ng/ml SCF. After 48 and 72 h, apoptosis was measured using the Pacific Blue Annexin V Apoptosis Detection Kit with 7-AAD. Flow cytometry measurements were performed with the FACS Gallios system (Beckman Coulter) and analyzed with FlowJo software taking together the values of both time points, as they did not differ. Apoptotic cells in histological sections were identified using TdT-mediated dUTP-biotin nick end labeling (TUNEL) according to the specifications of the manufacturer (Promega).