Cd29 apc

For the immunophenotypic characterisation of DPSCs (n = 3, passage four) and ADSCs (n = 3, passage four), commercial antibodies were used to analyse the expression of cell surface markers. The labelling was performed according to Rebelatto et al. [21 (link)]. Briefly, the cells were washed with PBS and incubated in the dark for 30 minutes with CD29-APC (1:20), CD14-FITC (1:20), CD45-FITC (1:20), CD19-FITC (1:20), CD34-APC (1:20), CD56-PE (1:10), CD105-APC (1:20), CD73-APC (1:33), CD90-PE (1:100), CD166-PerCP (1:33), annexin V (1:20) and 7-aminoactinomycin D (7-AAD)(1:20) (all markers and dyes used—Becton Dickinson, San Diego, CA, USA). The cells were washed with wash buffer and resuspended in a solution containing 1% formaldehyde. Isotypic IgG1 mouse antibodies were used as controls. Approximately 100,000 labelled cells were acquired by a FACSVerse flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and were analysed using FlowJo software with the default parameters (FlowJo, Ashland, OR, USA—version 8.1).

To explore the effect of Biodentine on the immunophenotype of LPS-treated macrophages, surface marker expression profile was evaluated by flow cytometry.
First, cells were harvested, collected, and stained with the following fluorinated antibodies: CD68-FITC (eBioscience, USA), CD14-PE-cy7 (BD Biosciences, USA), CD206-PE (BD Biosciences, USA), CD49F-PE (eBioscience, USA), CD29-APC (BD, USA), CD11b-PE (eBioscience, USA), HLA-DR-PerCPCy5.5 (BD Biosciences, USA), CD117-PE (BD Biosciences, USA), CD45-FITC (BD Biosciences, USA), and SSEA-FITC (BD Biosciences, USA), for 30 min at room temperature. Samples were acquired and analyzed with FACSDiva software version 8 on a FACSCanto II (BD Biosciences, USA) and FlowLogic software 7.3.

Immunofluorescence staining of cell surface antigens in MSC was performed by incubating cells for 30 min at 4 °C in the dark with mouse anti-human leukocyte antigen (HLA)-DR, DP, DQ (HLA class II)-FITC, HLA-ABC (HLA class I)-APC, CD34-FITC, CD44-FITC, CD105-PE, CD29-APC, and CD45-APC Abs (all from BD Biosciences, San Jose, CA,USA). Phenotypic characterization of macrophages generated by incubation with GM-CSF or M-CSF was assessed by staining with CD163-PE, CD197 (CCR7)-FITC, and CD80-APC (all from Miltenyi Biotec, Bergisch-Gladbach, Germany). Cells incubated in the absence of antibodies were used as controls. After incubation, cells were washed three times with PBS, fixed with 1% (w/v) formaldehyde in PBS, and analyzed by flow cytometry using a FACSCalibur analyzer and CellQuest software (both from BD Biosciences).

The immunophenotyping of hBM-MSC unlabeled and labeled with 50 µg Fe/mL of MFNP during 24 h (the maximum incubation time analyzed in this study) was performed using the following antibodies: CD19-FITC (clone:4G7) and CD34-PE (clone: 8G12) from BD Biosciences, San Jose, CA, USA; CD14-Alexa 700 (clone: M5E2), CD29-APC (clone: MAR04), CD35-FITC (clone: E11), CD44-PerCP-Cy5.5 (clone: G44-26), CD73-PE (clone: AD2), CD90-PE-Cy7 (clone: 5E10), HLA-DR-APC-H7 (clone: G46-6), CD106-FITC (clone: 51-10C9) all from BD Pharmingen, San Diego, CA, USA; CD31-V450 (clone: WM59), CD45-V500 (clone: H130), CD105-PE-CF546 (clone: 266) all from BD Horizon, San Jose, CA, USA, unstained samples and fluorescence minus one (FMO) was used as a fluorescence background control. Briefly, 1 × 10⁵ cells were incubated in the dark/room temperature for 30 min in the presence of antibodies at concentrations recommended by the manufacturers. Cells were then washed with a buffered solution, and at least 10,000 events were acquired using FACS LSRII FORTESSA equipment (BD Biosciences). Data analyses were performed using FlowJoTM software (BD Biosciences), and results were presented as graphics where the FMO is overlaid with the stained sample for each antibody/marker used.

Electroporated CD34⁺ HSPCs were stained with CD45-AlexaFluor700 (BioLegend, clone H130), CD34-PE/Cy7 (BioLegend, clone 581), CD44-BV421 (BioLegend, clone IM7), and CD29-APC (BD Pharmingen, clone MAR4 (RUO)). Mouse BM cells were stained with CD45-AlexaFluor700 (BioLegend, clone H130), HLA-A,B,C-PE/Cy7 (BioLegend, clone W6/32), CD33-PE (BioLegend, clone WM53), and CD19-BV605 (BD Biosciences, clone SJ25C1). All antibodies were used in the concentration recommended by the manufacturer. Dead cells and debris were excluded by forward scatter (FSC), side scatter (SSC), DAPI or 7-AAD staining (1:100 dilution prior to analysis). The flow cytometry analyses were performed with BD LSR II instrument (BD Biosciences) or BD LSRFortessa instrument (BD Biosciences). To analyse the data, the software FlowJo (FlowJo, LLC) was used.

In this work, we used rat and human bone marrow-derived mesenchymal stem cells (MSCs). Human MSCs 4–6 passage were taken from the culture bank of Research Institute of Clinical and Experimental Lymphology—Branch of the ICG SB RAS (RICEL–branch ICG SB RAS). Rat MSCs were harvested from the femoral and tibia bones of Wistar rats (8 weeks old) according to standard protocol. Cells in the fourth passage were used for phenotype screening of MSCs by flow cytometry (CytoFLEX S, Beckman Coulter, Brea, CA, USA) using antibodies to CD90FITS, CD90APC, CD29APC, CD73FITS (BD Biosciences, San Jose, CA, USA), and hematopoietic cell marker CD45FITS (BD Biosciences, San Jose, CA, USA).
All animal procedures were conducted in accordance with ethical standards of the international, institutional animal care guidelines. The Committee of Ethics in Animal Research of the Federal Research Center of Fundamental and Translational Medicine reviewed and approved all animal procedures (No. 10 of 26/03/2019).