Cd90 fitc

Phenotypical characterization of ASCs was performed using the BD FACSCalibur and analyzed with the CellQuest software (v. 1.0.1, Becton–Dickinson, Heidelberg, Germany). The cells were trypsinized, placed on ice for 30 min, and treated with the following labeled stemness-associated antigen markers: CD31-, CD34+, CD45–, CD54–, CD90+, CD105+, CD166+, HLA-ABC+, HLA-DR–. CD34-PE, CD90–FITC, and CD105-PerCP were part of the BD Stemflow Kit (Becton Dickinson, 562245). The other antibodies with the indicated specifications were purchased separately: CD31-FITC (R & D, Systems, Wiesbaden, Germany, FAB3567F), HLA-DR-PE (Becton Dickinson, 347401), CD166-PE (Becton Dickinson, 559263), CD34-APC (Becton Dickinson, 555824), CD54-FITC (Beckman Coulter, Krefeld, Germany, PN IM0726U), HLA-ABC-FITC (Becton Dickinson, 555552), and CD45-PE (Becton Dickinson, 555483). Table 1 shows the results from three donors. All experiments shown in this paper were performed with cells until reaching passage 5.

To examine surface markers, the cells were stained with monoclonal antibodies against cluster of differentiation (CD) 73 APC and CD90 FITC (Becton-Dickinson, New Jersey, USA), CD31 PE (Becton-Dickinson), CD45 PerCP (Becton-Dickinson), and human leukocyte antigen (HLA) class II (DR, DP, DQ) PE-Cy7 (Biolegend, San Diego, USA). The antibodies were added to the cells and incubated for 15 min at room temperature in the dark. The cells were washed and then resuspended in Phosphate Buffered Saline (PBS). Finally, the stained cells were analysed in a flow cytometer (FACSVerse, Becton Dickinson). FlowJo (Tree Star version 10.1r5 Inc, Ashland, USA) was used to analyse the data.

Evaluation of hPDLSCs phenotype was conducted by flow cytometry, as previously described [17 (link),19 (link)]. Briefly, 2.5 × 10⁵ cells were incubated for 30 min with following antibodies: anti-CD44-FITC, anti-CD105-FITC, anti-CD29-PE, and anti-CD45-FITC (Ancell Corporation, Bayport, MN, USA); anti-CD14-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany); OCT3/4-PE, CD73-PE, SOX2-Alexa488, SSEA4-FITC, CD90-FITC (Becton Dickinson, San Jose, CA, USA), and CD34-PE (Beckman Coulter, Fullerton, CA, USA). After incubation with appropriate secondary antibodies, fixation in 1 mL PBS 0.5% paraformaldehyde and washing, cells were analyzed using a FACStar-plus flow cytometry system and the FlowJo™ software v10.0.7 (TreeStar, Ashland, OR, USA).

Cells were rinsed twice with PBS, trypsinized and centrifugation at 200×g for 5 min, and then resuspended in 500 µL PBS. Approximately 5×10⁵ cells per 100 µL were labeled with primary mouse antibodies against rat CD29, CD34, CD44, CD45 and CD90 at 4°C for 30 min and washed. The labeled cells were analyzed using a flow cytometer (Beckton Dickinson, San Jose, CA, USA). The antibodies used in this experiment were: CD29- FITC, CD34-FITC, CD44- FITC, CD45- FITC and CD90- FITC (Beckton Dickinson, San Jose, CA, USA). Mouse IgG1-FITC (Beckton Dickinson, San Jose, CA, USA) was used as an isotype control.

The study of hPDLSC phenotype was performed by flow cytometry, as earlier stated (Diomede et al., 2018a (link)). Shortly, 2.5 × 10⁵ cells were incubated for 30 min with the following antibodies: anti-CD44-FITC, anti-CD105-FITC, and anti-CD29-PE (Ancell Corporation, Bayport, MN, United States); anti-CD14-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany); OCT3/4-PE, SOX2-Alexa488, CD73-PE,CD90-FITC (Becton Dickinson, San Jose, CA, United States) and CD34-PE (Beckman Coulter, Fullerton, CA, United States). After incubation with proper secondary antibodies, fixation in 1 mlL of PBS 0.5% paraformaldehyde and washing, cells were detected utilizing a FACStar -plus flow cytometry system and the FlowJo^TM software v10.0.7 (Tree Star, Ashland, OR, United States). The hPDLSCs at P2 were analyzed with an inverted light microscopy Leica DMIL (Leica Microsystem, Milan, Italy).

DPSC strains were characterized using flow cytometry. One hundred thousand cells (1 × 10⁵) from each population were used. The cells were stained with the following monoclonal antibodies: CD29-PE, CD31-FITC, CD34-FITC, CD44-PE, CD45-PE, CD73-FITC, CD90-FITC, CD105-PE and CD117-PE (Becton Dickinson, Franklin Lakes, NJ, USA). Appropriate isotype-matched control antibodies were used for all of the analyses. Cells were analyzed using a FACSCalibur flow cytometer with Cell Quest Software (Becton Dickinson).