Anti sca1 apc

Bone marrow stromal cultures derived from wild type and Sh3bp2^KI/KI mice were grown for 7 days in culture. On day 7 adherent cells were washed twice with PBS, digested with Accutase (Innovative Cell Technologies) for 5 minutes at 37°C, gently scraped and pipetted up and down to generate a single cell suspension. An equal volume of FACS staining media (1x HBSS, 10mM HEPES, 2% FCS, 2 mM EDTA, pH 7.4) was added and cells were centrifuged and resuspended in FACS staining buffer. Cell surface staining was carried out with anti-Sca1-APC and anti-CD45-FITC or anti-CD11B-APC and anti-CD45-FITC according to manufacturer’s recommendations (Miltenyi Biotech). FACS sorting was carried out on a Becton-Dickinson FACSARIA II (UCH Flow Cytometry Core).

On day 7 of culture, adherent cells were washed twice with PBS, digested with 5 mL of accutase (Innovative Cell Technologies) for 5 min at 37 °C, gently scraped and pipetted up and down to generate a single cell suspension. 10 mL of PBS was added to dilute accutase. Cells were then centrifuged and resuspended in FACS staining buffer (1× HBSS, 10 mM HEPES, 2% BSA (RNase free), 2 mM EDTA, pH 7.4). Cell surface staining was carried out with anti-Sca1-APC and anti-CD45-FITC according to manufacturer’s recommendations (Miltenyi Biotech). To a small volume of FACS staining buffer, Sytox blue was added to detect and remove dead cells during FACS, and SUPERase In RNase Inhibitor was added to prevent RNA degradation. Sorting was carried out on a Becton-Dickinson FACS-ARIA II (UCH Flow Cytometry Core). Two populations Sca1⁺CD45⁻ (mesenchymal stromal cells) and Sca1⁺CD45⁺ (hematopoietic cells) were collected into RNA protect obtained from Qiagen.