Cd105

Approximately 4 ×10⁵ hBMSCs at passage 3 were incubated with 1 ug fluorescein isothiocyanate-conjugated mouse anti-human monoclonal antibodies at room temperature for 45 min. After washing with fluorescence-activated cell sorting (FACS) buffer (PBS with 10% bovine serum albumin and 1% sodium azide) at 376 g for 5 min, the stained cells were suspended in 250 μl of ice-cold FACS buffer and then analyzed with FACS (BD Biosciences, USA). For each sample, 1 ×10⁴ events were counted. The percentage of positive signals was analyzed using technical flow cytometry. The antibodies, including CD-29 (#555443; BD Pharmingen), CD-34 (#555822; BD Pharmingen), CD-44 (#555478; BD Pharmingen), CD-45 (#566156; BD Pharmingen), CD-90 (#551401; BD Pharmingen), and CD-105 (#323208; Biolegend), were used in this study.

Cells were detached from culture dish using HyQtase and blocked with Fc (CD32/16) at RT for 15 min, followed by incubating with primary antibodies conjugated with fluorescence at 4 °C for 30 min in darkness [37 (link)]. The cells were then assessed using BD FACS Canto II, and the data was analyzed using FlowJo software (TreeStar). Analyses included CD117 (Abcam, Cat. ab5506, 1:10), Sca1 (BioLegend, Cat. 108111, 1:50), CD73 (BioLegend, Cat. 127209, 1:50), CD90.2 (BioLegend, Cat. 105307, 1:50), CD105 (BioLegend, Cat. 120407, 1:50), CD34 (BioLegend, Cat. 128611, 1:50), CD31 (BioLegend, Cat. 102407, 1:50), CD45 (BioLegend, Cat. 103111, 1:50), CD11b (BioLegend, Cat. 101207, 1:50), MHC I (eBioscience, Cat. 17-5957-80, 1:50), and MHC II (eBioscience, Cat. 17-5957-82, 1:50).

Hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) enzyme activity kits were obtained from Jiancheng (Nanjing, China). MitoTracker Red CMXRos (M7512) and Alexa Fluor 488 Phalloidin (A12379) were obtained from Invitrogen (Carlsbad, CA, USA). The XF Cell Energy Phenotype Test Kit, XFp extracellular flux analyzer, XFp culture microplates, and bicarbonate-free DMEM were obtained from Seahorse Bioscience Agilent Technologies (North Billerica, MA, USA). Hoechst 33342 (B2261) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP, C2759) were purchased from Sigma–Aldrich (St Louis, MO, USA). The following antibodies were used: CD90 (FITC, mouse anti-human, BioLegend, 328107), CD105 (PerCP/Cy5.5, mouse anti-human, BioLegend, 323215), CD19 (PE, mouse anti-human, BioLegend, 982402), CD34 (PE, mouse anti-human, BioLegend, 343505), AMPK (Abcam, ab80039), p-AMPK (Abcam, ab133448), anti-GAPDH (Abcam, ab9485,ab8245), goat anti-rabbit IgG (H + L)-HRP (Bio-Rad Laboratories, 1706515), goat anti-mouse IgG (H + L)-HRP (Bio-Rad Laboratories,1706516) and goat anti-rabbit IgG (H+L) Alexa Fluor 594-conjugated secondary antibody (Invitrogen, R37117).

Culture plates were trypsinized, inactivated, and supplied with DMEM/F-12 before being centrifuged at 1300 rpm for 5 min, and the supernatant was discarded. Conjugated primary antibodies were added to the pellet after it had been resuspended in PBS with 1 × 10⁶ cells per tube. Mesenchymal cells were identified using the markers CD105+ (Biolegend, San Diego, CA, USA 323218), CD73+ (BD Biosciences Franklin Lakes, NJ, USA, 563198), CD90+ (BD Biosciences, San Diego, CA USA 555597), CD45- (MACSMylteny Biotec San Diego, CA, USA 130-080-202), CD14- (BD Biosciences, San Diego, CA USA 561712), CD19- (BD Biosciences, San Diego, CA USA 560911), and CD34- (BD Biosciences San Diego, CA USA 562577). The information was recorded using a Fortessa flow cytometer (BD) and examined using the Flowjo 7.6 application.

PDLSCs were labelled with antibodies against mesenchymal cell surface markers and analysed using flow cytometry, as described previously.15 Briefly, to immunophenotype the PDLSCs, 1 × 10⁵ cells at the third passage were incubated with phycoerythrin‐conjugated or fluorescein isothiocyanate‐conjugated mouse monoclonal antibodies against human Stro‐1 (Abcam), CD146 (eBioscience), CD105 (BioLegend), CD29 (Abcam), CD166 (BioLegend), CD31 (eBioscience), CD14 (Boster) and CD45 (eBioscience) per the manufacturers’ instructions. Antibody labelling was conducted at 4°C in the dark for 1 hour. A cell suspension without antibodies served as the blank control. The cells were subjected to flow cytometric analysis using a flow cytometer (BD FACSCalibur; BD Biosciences). Data were analysed with the BD CellQuest Pro Software (BD Biosciences), and representative results from one of three independent experiments are shown.