Pe conjugated anti sca1

The monoclonal antibodies used in this study are listed below. The purified anti-mouse TER-119, Gr-1, B220, CD3 and PCNA and PE-conjugated anti-mouse TER-119 antibodies were purchased from BD Pharmingen (San Diego, CA, USA). APC-conjugated CD71, PE-conjugated anti-Sca1 and APC-conjugated anti-c-kit antibodies were purchased from Biolegend (San Diego, CA, USA). Biotinylated anti-c-kit antibody was purchased from Abcam (Cambridge, UK). Normal rabbit IgG, biotinylated goat anti-rat IgG, biotinylated goat anti-mouse IgG, biotinylated goat anti-hamster IgG, Texas Red-conjugated goat anti-Rat IgG antibodies and an avidin-biotin complex kit (ABC Elite standard kit) were purchased from Vector Laboratories (Burlingame, CA, USA). The anti-mouse F4/80 antibody was obtained from AbD Serotec (Kidington, UK). The Lineage Cell Depletion kit was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

For the analysis or sorting of GFP⁺CD45⁻ or CD105⁺CD90⁺CD45⁻CD11b⁻ cells in peripheral circulation, blood samples were collected from mice by intracardiac puncture. After the process of red blood cell lysis with commercial ammonium‐chloride‐potassium lysis buffer (Quality Biological, Gaithersburg, MD, http://www.qualitybiological.com), cells were washed and counted. We incubated equal numbers of cells for 45 minutes at 4°C with fluorescein isothiocyanate (FITC)‐conjugated anti‐GFP (PA1‐46326; Thermo Fisher Scientific, Waltham, MA, https://www.thermofisher.com) and peridinin chlorophyll protein (perCP)‐conjugated anti‐CD45 (103132; BioLegend, San Diego, CA, http://www.biolegend.com). We also incubated cells with phycoerythrin (PE)‐conjugated anti‐Sca1 (108108; BioLegend), allophycocyanin‐conjugated anti‐CD90 (140311; BioLegend), perCP‐conjugated anti‐CD45 (103132, BioLegend), and FITC‐conjugated anti‐CD11b (101208, BioLegend) antibodies. Probes were analyzed using Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, http://www.bd.com) and CellQuest software.