Cd44 fitc

ADSCs from passage 1 (P1) were cryopreserved by standard protocol in 10% dimethyl sulfoxide (DSMO, Sigma Aldrich, cat. no. C6295) and kept at −80°C. After thawing, the cells were grown one passage. P2 cells were harvested and analysed for the expression of surface antigens by staining with CD29-APC (BioLegend, cat. no. 303007, clone TS2/16), CD45-PE (Bio-Rad, cat. no. MCA2220PE, clone 1.11.32), CD44-FITC (Bio-Rad, cat. no. MCA2219F, clone 25.32), or CD31-FITC (Bio-Rad, cat. no. MCA1097F, clone CO.3E1D4) for FC analysis on a FACSVerse (BD Biosciences). Corresponding isotype antibodies from BD Pharmingen were used as negative controls. In brief, cells were detached by EDTA, washed with PBS, counted, and stained with fixable viability dye eFluor 506 (eBioscience, cat. no. 65-0866-14) to show live/dead cells. The cells were subsequently fixed by cytoperm/cytofix (BD, cat. no. 51-2090KZ). 5·10⁵ cells in each sample were washed with PBS and stained with relevant antibodies for 30 min at 4°C in the dark. The cells were stained for CD45 and CD29 in addition to either CD44-FITC or CD31-FITC, and another tube was stained with the isotype controls. 5–10 μl antibody solution per 200 μl cell suspension was used according to the manufacturer's recommendations.

For flow cytometry analysis, oBMSC were stained with the following anti-human antibodies: CD34-APC, CD90-FITC, CD73-APC, CD105-PE, CD146-APC, CD271-PE, HLA-DR-PE, and isotype controls (Miltenyi Biotec). Additionally, the following anti-sheep antibodies were used: CD45-FITC and CD44-FITC (Bio-Rad). Cells were stained as per the manufacturers’ instructions, and analysis was completed using an LSR II flow cytometer (BD Biosciences). In this analysis, we also included sheep bone marrow mononuclear cells (MNC) and hBMSC. These cell populations served as controls for antibody performance against a common hBMSC population, and for sheep MNC, where haematopoietic cells (CD45⁺) would be expected. Data were analysed using FlowJo software, version 10 (BD Biosciences).

AdMSCs at passage 3 were washed three times with sterile PBS. Cells were incubated with 3 ml of Cellstripper (Corning) for up to 15 min, tapping on plate every 5 min to dislodge cells. Cells were scraped and pooled, then centrifuged for 5 min at 1000×g at 4C. Cells were resuspended in FACS buffer (1% hiFBS and 1 mM EDTA in sterile PBS) and centrifuged at 1000×g for an additional 3 min. The cell pellet was resuspended in FACS buffer and aliquoted into 1.5 ml tubes. The cells were incubated for 30 min on ice with following antibodies: Mouse IgG FITC (1:2000, Bio-Rad), CD34-FITC (1:1000, Bio-Rad), CD44-FITC (1:1000, Bio-Rad), CD45-FITC (1:1000, Bio-Rad), CD73-FITC (1:2000, BioLegend), CD90-FITC (1:100, Abcam), CD105 (1:100, Bio-Rad, with anti-rat 488 secondary, 1:500). Cells were centrifuged at 500×g for 30 s and washed three times with FACS buffer. On the final wash, cells were transferred into library tubes with 500 µl FACS buffer and placed on ice. Live/dead stain (Sytox AADvance, Invitrogen) was added at 1:100 dilution to all samples excluding reference controls. 10,000 live cells per sample were analyzed using the Cytek™ 4-laser Aurora Cytometer and data was processed in FlowJo, gating on single cells, live cells, then FITC-positive and FITC-negative cells.

hUC-MSC-p3 and hUC-MSC-p15 were collected and treated with 0.25% trypsin. The cells were individually stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated anti-marker monoclonal antibodies in 100 μl PBS for 15 min at room temperature, or for 30 min at 4°C, as recommended by the manufacturer. The antibodies used were specific for the following human antigens: CD34-PE, CD44-FITC, CD45-PE, CD73-PE, CD90-PE and CD105-PE (10 μl for 1×10⁶ cells; AbD Serotec, Raleigh, NC, USA). Cells were analyzed on a Cytometer 1.0, Cytomics™ FC500 flow cytometry system (Beckman Coulter, Brea, CA, USA). Positive cells were counted and the signals for the corresponding immunoglobulin isotypes were compared (15 (link)).

Cells were trypsinized at passage 2. Subsequently, 1 × 10⁵ cells per sample were washed with PBS supplemented with 2% FCS. The flow cytometry analysis utilized the following monoclonal antibodies and their respective isotype controls: PE-CD29 (Clone TS2/16, Mouse IgG, 1:50, Biolegend, San Diego, CA, USA), FITC-CD31 (Clone CO.3E1D4, IgG2a, 1:50, Biorad, Hercules, CA, USA), FITC-CD44 (Clone 25.32, IgG, 1:50, Biorad), Purified CD90 (Clone DH24A, IgM, 1:50, Monoclonal Antibody Center), FITC-PanB cells (Clone CVS36, IgG1, 1:50, Biorad). A total of 1 × 10⁴ events were measured per sample.

For the flow cytometry, cells were trypsinised at passage 2, and 1 × 10⁵ cells per sample were washed with PBS +/+ supplemented with 2% FSC (Sigma Aldrich, F7524). The following monoclonal antibodies and respective isotype controls were used for the flow cytometry: PE-CD29 (Clone TS2/16, Mouse IgG, 1:50, Biolegend, San Diego, CA, USA), FITC-CD31 (Clone CO.3E1D4, IgG2a, 1:50, Biorad, Hercules, CA, USA), FITC-CD44 (Clone 25.32, IgG, 1:50, Biorad), Purified CD90 (Clone DH24A, IgM, 1:50, Monoclonal Antibody Center), FITC-PanB cells (Clone CVS36, IgG1, 1:50, Biorad). A total of 1 × 10⁴ events were measured per sample.