Anti cd105

Human abdominal subcutaneous adipose tissues were harvested during liposuction procedures after obtaining signed informed consent in the Department of Plastic Surgery, Affiliated Hospital of Zunyi Medical University, China. ADSC isolation and culture was performed as previously described.¹⁵ Briefly, human adipose tissue was minced and digested with 0.075% collagenase type I (Sigma) for 45 minutes at 37°C. After digestion, an equal volume of Dulbecco's modified Eagle's medium (DMEM; Gibco, Carlsbad, California) supplemented with 10% fetal bovine serum (FBS; Gibco) was added and the suspension was passed through a 200 μm mesh filter followed by centrifugation at 800g for 5 minutes). The stromal‐vascular fraction cell pellets were resuspended and cultured at 37°C in 5% CO₂ in DMEM supplemented with 10% FBS and 1% penicillin‐streptomycin (Gibco). ADSCs were subcultured at 80% confluence, and passage‐3 cells were used in the study procedures. ADSCs surface marker expression was assayed by flow cytometry. Suspensions of 1 × 10⁶ ADSCs were incubated with anti‐CD90, anti‐CD73, anti‐ CD105, anti‐CD34, anti‐CD11b, anti‐CD19, anti‐CD45, or anti‐HLA‐DR antibodies (1 mg/mL; Abcam) at room temperature for 30 minutes, washed with phosphate‐buffered saline (PBS), and analyzed with a MoFlo XDP flow cytometer (Beckman Coulter, Brea, California) and Kaluza software (Beckman Coulter).

Anti-CD9, anti-CD63, anti-Alix, anti-calnexin, anti-CD31, anti-CD34, anti-CD45, anti-CD73, Anti-CD90, anti-CD105, anti-Collagen I, and anti-Fibronectin antibodies were purchased from Abcam (Cambridge, UK). Anti-Ki-67, anti-RhoA, anti-Rac1, anti-Cdc42, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies were purchased from Proteintech (Rosemont, IL, USA). IRDye 800cw-conjugated goat anti-rabbit/anti-mouse IgG secondary antibodies were purchased from Abbkine (Redlands, CA, USA). PKH-26 and PKH-67 kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine™ RNAiMAX, FM™ 4-64FX, and ActinGreen™ 488 were purchased from Thermo Fisher (Eugene, Oregon, USA). SiCdc42, Cdc42-EGFP, and Cdc42-mCherry fusion protein expression plasmids were purchased from GenePharma (Suzhou, China). The Cdc42 inhibitor ML141 was purchased from MedChemExpress (Monmouth Junction, USA).

pMSC were characterised by flow cytometry using anti-porcine specific or cross-reactive antibodies as previously described [28 (link)]. In particular, anti-CD45 (LifeSpan Biosciences, Seattle, Washington), CD29 (Acris Antibodies, Herford, Germany), anti-CD90, anti-CD11b and anti-CD105 (all from Abcam, Cambridge, UK) were used. Cells were tested for their ability to differentiate into osteoblasts and adipocytes at early passages (P3), as previously described [28 (link)].

A flow cytometric assay of cell surface marker expression was conducted in 1 × 10⁶ ASCs that had been stained with anti-CD90, anti-CD73, anti-CD105, anti-CD34, anti-CD11b, anti-CD19, anti-CD45, and anti-HLADR antibodies (1 mg/ml; Abcam, Cambridge, UK) and suspended in PBS. The samples were incubated for 30 minutes at room temperature, washed with PBS, and then analyzed with a MoFlo XDP flow cytometer (Beckman Coulter, Brea, CA) and Kaluza software (Beckman Coulter).

Cultured ADSCs at passage three were characterized by flow cytometric evaluation of the expression of MSCs markers (CD29, CD44, CD105) with negative expression of hematopoietic and endothelial markers (CD45, CD34, CD31), this was performed following previously published protocols [24 (link),30 (link)]. Briefly, ADSCs were detached, centrifuged to pellet the cells, washed twice, and fixed with 10% neutral buffered formalin for 30 min, washed again, and finally the cell pellet was resuspended in 100 µL FACS buffer containing primary antibody for 60 min at room temperature in the dark. The primary antibodies are FITC-labeled anti-CD29 (BioLegend, San Diego, CA, USA), anti-CD44 (BD Pharmingen, San Diego, CA, USA), anti-CD105 (abcam, San Diego, CA, USA), anti-CD45 (BioLegend, San Diego, CA, USA), anti-CD34 (BD Pharmingen, San Diego, CA, USA), and anti-CD31 (BD Pharmingen, San Diego, CA, USA). Then, propidium iodide was added to exclude dead cells, and the data was obtained on an Attune NxT Flow Cytometer (Applied Biosystems, Waltham, MA, USA). Twenty-thousand events were acquired, and the data was analyzed by Attune NxT Software v 3.1 (Applied Biosystems, Waltham, MA, USA).

PDLSCs were cultured on 24-well plates with glass coverslips at a density of 10⁵ cells/well overnight. Then, PDLSCs were fixed with 4% paraformaldehyde and incubated with anti-CD146 (1:100; Abcam, USA), anti-CD105 (1:100; Abcam, USA), and anti-CD45 (1:200; Abcam, USA) primary antibodies. The samples were then treated with rhodamine/FITC-conjugated secondary antibodies (1:1000; Sigma-Aldrich, USA) and stained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich, USA). Jurkat cells were stained with anti-CD45 antibody as a positive control. The images were captured with a fluorescence microscopy (OLYMPUS, Japan).