Anti cd90 fitc

In in vivo experiments, mice were sacrificed after tMCAO. The BALF was centrifuged at 500 g to collect cells. After being washed with PBS, cells were fixed and permeabilized (Invitrogen, Intracellular Fixation & Permeabilization Buffer Set), then stained with intracellular antibodies. The following antibodies were used: anti‐CD45‐BV421 (Biolegend, 103 134, clone: 30‐F11, 1:400), anti‐ F4/80‐AF488 (Biolegend, 123 120, clone: BM8, 1:400), anti‐LY6G‐APCCy7 (Biolegend, 108 424, clone: RB6‐8C5, 1:400), anti‐CD11c‐APC (Biolegend, 117 310, clone: N418, 1:400), anti‐CD3‐PECy7 (Biolegend, 100 220, clone: 17A2, 1:400), anti‐CD19‐PE (Biolegend, 152 408, clone: 1D3/CD19, 1:400), anti‐CD45‐PerCPCy5.5 (Biolegend, 368 504, clone: 2D1, 1:400), anti‐CD90‐FITC (Biolegend, 328 108, clone: 5E10, 1:400), anti‐CD34‐APC (Biolegend, 343 509, clone: 581, 1:400), and anti‐CD29‐PE (Biolegend, 303 004, clone: TS2/16, 1:400). Cell viability of BMDM was assessed using the Annexin V‐APC/PI apoptosis Detection Kit (KeyGEN, KGA1030‐100) according to manufacturer' s instructions.

To characterize the expression of surface markers, the MSCs were immune-stained and detected by flow cytometry analysis. Antibodies used in this detection are anti-CD73-PE, anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD34-FITC, anti-CD19-PE, anti-CD45-APC and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA).
To assess the apoptotic rates, H9c2 cells were seeded in a 6-well plate at 30,000 cells/well for 24 h and then desired treatments were performed for 24 h. Cells were stained using an Annexin V/PI staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions and detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Flow cytometry was used to observe the ADSC phenotypes. Passage 3 ADSCs were trypsinized and incubated with anti-CD34-PE, anti-CD44-PE, anti-CD73-FITC, anti-CD90-FITC, and anti-HLA-DR-PE (Biolegend, San Diego, CA, USA). After three washes with PBS, the fluorescence of ADSCs was observed. To verify adipogenic differentiation and osteogenic differentiation, ADSCs at passage 3 were incubated separately with adipogenic and osteogenic differentiation medium (Gibco, United States) following the supplier’s instructions for 2 or 3 weeks. The induced cells were stained with Oil Red O and Alizarin Red respectively and microscopically imaged.

Cells from mouse synovium were obtained as described above for cell culture, with the addition of DNase I (1 mg/ml; Sigma #DN25) during collagenase digestion. Cells were then resuspended in red cell lysis buffer for 3 min at room temperature, and red cell lysis was stopped by adding 20 ml of cold PBS. Cells were then centrifuged and stained with flexible viability dye eFluor 780 (Invitrogen #65-0865-14) at 1 μg/ml in PBS for 20 min on ice to discriminate live and dead cells. FC receptor was blocked using CD16/CD32 specific antibody (Invitrogen, #14-0161-85) for 20 min on ice. Cells were then incubated with primary antibodies or isotype controls at 1 μg/ml in FACs buffer (PBS 1%FBS 2 mM EDTA) for 20 min at 4°C. Antibodies used were: anti-CD31PE (Invitrogen, #12-0311-81), anti-CD45-PE (Biolegend, #103106), anti-CD90-FITC (Biolegend, #105316), anti-PDPN-A647 (Biolegend, #156204), anti-rat IgG2b-PE (BD bioscience #25393), anti-rat IgG2a-PE (Biolgend, # 400508), anti-rat IgG2b-FITC (Biolegend #400634) and anti-rat IgG2a-APC (Biolegend, #400512). Cell sorting was performed using FACS Aria III or FACS Aria IIu (all from BD), data were analyzed with FlowJo software 10.7.1.

Human PDGFRα + cMSCs were isolated by explant culture and purified by fluorescence-activated cell sorting as previously described^{17 (link)}. Briefly, frozen left ventricular samples (~100–200 mg) were removed from liquid nitrogen and quickly thawed by washing twice with ice cold DPBS solution. The tissue was minced into small segments and then placed in 6-well plates coated with 0.1% (wt/vol) gelatin. All explants were cultured in MEMα medium (Sigma-Aldrich) supplemented with 20% FBS (Sigma-Aldrich), 2 mM L-glutamine and 1x penicillin-streptomycin (Sigma-Aldrich), in a 5% CO₂ humidified incubator at 37 °C. The culture media was replaced every 2–3 days. After 2–3 weeks, the outgrowth of cells was dissociated with trypsin-EDTA (Sigma-Aldrich) and stained with anti-PDGFRα antibody-APC (R&D; 1:10), anti-CD31-PE (R&D; 1:20) and anti-CD90-FITC (BioLegend; 1:20). The cells were then sorted for PDGFRα+/CD90+/CD31⁻ fraction using Influx machine (BD Biosciences), as described previously^{17 (link)}.

Human adipose tissues and foreskins were obtained in accordance with procedures approved by the Ethics Committee at the Tongji Medical Collage of Huazhong University of Science and Technology. Primary fibroblasts were isolated from foreskins derived from routine pediatric circumcisions, using previously described protocols [26 (link)]. ADSCs were isolated and cultured in accordance with our established protocol [27 (link)]; ADSCs at passages 3–8 were used for experiments. Flow cytometry was conducted to identify ADSC phenotypes. ADSCs were incubated for 1 h with anti-CD34-BV421, anti-CD44-APC, anti-CD105-PE, anti-CD73-FITC, anti-CD31-FITC, and anti-CD90-FITC antibodies (all from Biolegend, San Diego, CA, USA); fluorescence was observed thereafter. For multi-lineage differentiation potential assays, ADSCs were incubated separately with adipogenic medium and osteogenic differentiation medium (Cyagen Biosciences Inc., Guangzhou, China). Cells were stained with Oil Red O and Alizarin Red, respectively. Subsequently, the stained cells were imaged using a microscope.

Approximately 1 × 10⁶ PDLSCs at the third passage were added in blocking buffer and incubated with anti-STRO-1 immunoglobulin (Ig)M (Santa Cruz, CA, USA) for 1 h at 37°C, then incubated with goat anti-mouse IgM FITC secondary antibodies (Molecular Probes, Eugene, Carlsbad, CA, USA) for an additional 1 h. PDLSCs incubated with the secondary antibody only was used as a negative control. Another 1 × 10⁶ PDLSC was incubated with anti-CD146-PE, anti-CD45-FITC, and anti-CD90-APC antibodies (BD Biosciences, USA), at 37°C for 30 min. 1 × 10⁶ BMSCs at the third passage were incubated with antibodies at 37°C for 30 min, including anti-CD29-FITC, anti-CD 45-FITC, anti-CD44-FITC, anti-CD11b/c-FITC, and anti-CD90-FITC (BioLegend, CA, USA). The cells were then analyzed by flow cytometry (Attune NxT, Invitrogen, USA) and Treestar FlowJo software.