Ter119 pecy7

In the experimental group, BMCs were obtained from murine femurs and tibias 1 week after preparation of periodontal defects. Untreated mice were used as controls. To evaluate the ratio of bone marrow MSCs for all collected cell counts, we conducted at least three experiments using two mice in the experimental and control groups. BMCs were obtained as described previously (Morikawa et al., 2009 (link)). Briefly, femurs and tibias were aseptically removed from two mice and bones were crushed with an ice-cold pestle and mortar. Bone chips, including marrow were rinsed with HBSS+ and were digested using collagenase (#032-22364; Wako) for an hour at 37°C. Collected BMCs were hemolyzed and FcR was blocked with anti-mouse CD16/32 (#553142; BD) on ice for 5 min. BMCs (2–5 × 10⁵) were multi-stained with CD45.2-APC-eFlour780 (#47-0454; eBioscience, San Diego, CA, USA), TER119-PECy7 (#25-5921; eBioscience), pdgfrα-PE (#12-1401-81; eBioscience), Sca-1-APC (#17-5981-81; eBioscience) (1: 100 dilution, 30 min, on ice) and 7AAD (#559925; BD) (1: 100 dilution, 10 min, on ice). The ratio of MSCs [CD45.2 (−), TER119 (−), 7AAD (−), pdgfrα (+), Sca-1 (+)] in BMCs was evaluated by FACS analysis.

For Sc-FACS-Seq analysis of GFP⁺ cells, BMCs were prepared from the hind limbs of P21 Cko mice (see above) and GFP⁺ cells were FACS sorted using a BD FACSAria II (Becton Dickinson) FACS sorter into a sterile 96-well plate at a density of one cell/well containing 10X lysis buffer (Clontech, # 635013) with Protector RNAse inhibitor (Roche). The plates were frozen immediately on dry-ice and stored at −80°C until ready to process for RNA-Seq. Two 96 well plates of GFP⁺ sorted single cells (GFP-Cko1, GFP-Cko2) were carried forward for analysis with each plate receiving cells sorted from a pool of BMCs made from two Cko mice. Thus, a total of four Cko mice were analyzed. For RNA-Seq analysis of skeletal stem cells (SSCs) defined as CD45⁻, Ter-119⁻, CD31⁻, CD105⁺ cells (Patra et al., 2018 (link)), 10⁶ cells from WT and Cko mice were stained as reported previously with fluorescent-tagged antibodies to CD45 (PE-Cy7) (BD Pharmingen, 552848), Ter-119 (PE-Cy7) (eBioscience, 25-5921), CD31 (BV421) (BD Pharmingen, 563356), and CD105 (Alexa-fluor 647) (BD Pharmingen, 562761), washed to remove unincorporated antibodies, and SSCs were FACS sorted at a density of one cell/well into a sterile 96-well plate containing lysis buffer. A total of two WT (SSC-WT1, SSC-WT2) and two Cko (SSC-Cko1, SSC-Cko2) mice were analyzed by RNA-Seq.