BMSCs and AMSCs were harvested for 5 min at 37 °C, plated by 200 × g centrifugation for 5 min, and rinsed with chilled PBS. Next, 5 µl of fluorescein isothiocyanat (FITC)-conjugated antibodies, including CD34-PE, CD90-FITC, CD45-FITC, and CD73-FITC (Thermo Fisher Scientific, Germany), was added to the BMSCs and AMSCs and then stored at room temperature (RT) and a dark place for 20 min. The samples were evaluated with a flow cytometer (BD FACS Caliber; San Jose, CA, USA) [33 (link), 34 (link)].
Cd90 fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD90-FITC is a fluorescently labeled antibody that binds to the CD90 (Thy-1) cell surface antigen. It is commonly used in flow cytometry applications to identify and characterize cell populations expressing the CD90 marker.
Automatically generated - may contain errors
Lab products found in correlation
Cd73 apc, by Thermo Fisher Scientific (11 mentions)
Cd105 pe, by Thermo Fisher Scientific (10 mentions)
Cd34 pe, by Thermo Fisher Scientific (9 mentions)
Cd45 fitc, by Thermo Fisher Scientific (8 mentions)
Cd105 apc, by Thermo Fisher Scientific (8 mentions)
Cd73 fitc, by Thermo Fisher Scientific (7 mentions)
Cd34 fitc, by Thermo Fisher Scientific (5 mentions)
Cd73 pe, by Thermo Fisher Scientific (5 mentions)
Cd29 fitc, by Thermo Fisher Scientific (5 mentions)
Cd45 fitc, by BioLegend (5 mentions)
Cd45 pe, by Thermo Fisher Scientific (4 mentions)
Cd105 pe cy7, by Thermo Fisher Scientific (4 mentions)
Hla dr fitc, by Thermo Fisher Scientific (4 mentions)
Accuri c6 flow cytometer, by BD (4 mentions)
Facscalibur system, by Beckman Coulter (4 mentions)
Hla dr pe cy5, by BioLegend (4 mentions)
Cd29 pe, by Thermo Fisher Scientific (4 mentions)
Cd31 fitc, by Thermo Fisher Scientific (3 mentions)
Cd34 percp, by Thermo Fisher Scientific (3 mentions)
Cd44 fitc, by Thermo Fisher Scientific (3 mentions)
38 protocols using cd90 fitc
1
Multilineage Stem Cell Characterization
2
Purification and Characterization of CD34+ Cells
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Unmanipulated UCB, BM or mobilized PB CD34+ cells or cells from cultures treated with cytokines alone or cultures treated with VPA were stained with anti-human CD34-APC (BD Pharmingen), CD90-FITC (Thermo Fisher Scientific), CD201-PE (Bio-Legend), CD45RAef-506 (Thermo Fisher Scientific), CD38 SB-702 (Thermo Fisher Scientific) for 30 min at 4°C, washed and analyzed using an Attune flow cytometer (Thermo Fisher Scientific). Analyses were performed with Attune software (Thermo Fisher Scientific) and FlowJo Software (BD Biosciences). Compensation parameters are assessed by using AbCTM Total Antibody Compensation Bead Kit (Thermo Fisher Scientific) according to the manufacturer instructions. Doublet exclusion was performed by FSC-H/FSC-A selection followed by SSC-H/SSC-A selection. Gating of positive/negative population was performed by using fluorescence minus one control (FMO) strategy (Papa et al., 2020b (link)). Purification of the various CD34+ cell subpopulations is performed in media supplemented with cytokines or cytokines and VPA using a BD FACSAria (BD Bioscience).
3
Multiparametric Flow Cytometry Characterization
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The cells (1x106 per group) were resuspended in 100 µl of flow buffer (cat. no. PAB180076; Bioswamp Wuhan Beinle Biotechnology Co., Ltd.) in an Eppendorf tube and 2 µl of CD45-FITC (cat. no. 11-9459-42, eBioscience; Thermo Fisher Scientific, Inc.), CD34-FITC (cat. no. CD34-581-01; Invitrogen; Thermo Fisher Scientific, Inc.), CD73-FITC (cat. no. 11-0739-42r; eBioscience; Thermo Fisher Scientific, Inc.), CD90-FITC (cat. no. 11-0903-82; eBioscience; Thermo Fisher Scientific, Inc.) or CD105-FITC (cat. no. MA1-19594; Invitrogen; Thermo Fisher Scientific, Inc.) was added. The cells were incubated in the dark for 45 min at 4˚C, following which, 400 µl of flow cytometry dyeing buffer (cat. no. PAB180076; Wuhan Beinlai Biotechnology Co., Ltd.) was added to each tube. The cells were subjected to flow cytometry (CytoFLEX S; Beckman Coulter, Inc.) and the results were analyzed using the CYEXPERT software (CXP Analysis 2.0; Beckman Coulter, Inc.).
Cells were cultured for 24 h at 37˚C, harvested, treated with 1 ml of pre-cooled PBS and centrifuged at 1,000 x g for 5 min at 4˚C. Subsequently, 10 µl of Annexin V-FITC and 10 µl of PI were added. The cell samples were then analyzed using flow cytometry as aforementioned. A one-step fluorescence compensation strategy was used to eliminate interference with the FITC channel (21 (link)).
Cells were cultured for 24 h at 37˚C, harvested, treated with 1 ml of pre-cooled PBS and centrifuged at 1,000 x g for 5 min at 4˚C. Subsequently, 10 µl of Annexin V-FITC and 10 µl of PI were added. The cell samples were then analyzed using flow cytometry as aforementioned. A one-step fluorescence compensation strategy was used to eliminate interference with the FITC channel (21 (link)).
4
Flow Cytometry Analysis of Mesenchymal Stem Cells
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Cells were harvested and the cell pellet was resuspended in PBS to 106 cells/100 μl. The cell suspension was incubated with the following antibodies: HLA-DR-APC, CD34-FITC, CD45-PE, CD73-PE, CD90-FITC, and CD105-FITC (eBioscience). An isotype control antibody (eBioscience) was used for each. Approximately 5 μl of each antibody (1:10 dilution, according to the antibody datasheets) per tube was added, and this was incubated for 15 min in the dark at 2–8 °C. Cells were completely washed with PBS and resuspended in 1% (w/v) paraformaldehyde (Sangon Biotech, China). Samples were subjected to flow cytometry (BD Biosciences, USA) and the data were analyzed by FlowJo software (FlowJo LLC, Ashland, OR, USA).
5
Isolation and Characterization of Rat MSCs
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MSCs were isolated from the bone marrow of 4-week-old rats. The experimental rats were euthanized via an intraperitoneal injection of sodium pentobarbital, and the femur and tibia were separated under sterile conditions.10 (link) The marrow cavity was washed repeatedly with Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO, Grand Island, NY, USA). The cell suspension was centrifuged at 350 g for 10 min, and the supernatant was discarded. The precipitate was mixed with erythrolysis buffer (Solarbio, Beijing, China) for 3 min. Cells were subjected to centrifugation and washing, followed by culture in DMEM supplemented with 10% fetal bovine serum.
The specific antibody markers of MSCs were detected using flow cytometry. Positive antibody markers included CD90-FITC (eBioscience, San Diego, CA, USA) and CD105-FITC (Abcam, Cambridge, UK). Negative markers were CD34 (Abcam) combined with goat anti-rabbit IgG (Abcam) labeled with phycoerythrin and FITC-CD45. Cells stained with an isotype control (IgGFITC) were used as the negative control.
The specific antibody markers of MSCs were detected using flow cytometry. Positive antibody markers included CD90-FITC (eBioscience, San Diego, CA, USA) and CD105-FITC (Abcam, Cambridge, UK). Negative markers were CD34 (Abcam) combined with goat anti-rabbit IgG (Abcam) labeled with phycoerythrin and FITC-CD45. Cells stained with an isotype control (IgGFITC) were used as the negative control.
6
Immunophenotypic Characterization of MSCs
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The immunophenotype of MSCs was determined by flow cytometry according to previous described methods29 (link),30 (link). Second-passage MSCs were harvested, non specific binding was blocked and the cells were stained with different antibodies against the human antigens: CD29-FITC, CD90- FITC and CD45-FITC(eBioscience, California, USA). Nonspecific isotype matched antibodies were used as controls. The fluorescence intensity of the cells was evaluated by flow cytometer and the data were analyzed with the CytExpert software (Beckman Coulter, California, USA). Results are presented as percent positively stained cells.
7
Isolation and Characterization of Placental MSCs
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Human placental tissues were obtained from healthy delivering women in accordance with the Austrian Hospital Act (KAG 1982) after written informed consent and the study was approved by the Ethic Commission of Lower Austria (GSl-EK-4/3122015). Amnion-derived MSCs from placental tissue were isolated and characterized with CD73-APC, CD90-FITC and CD105-PE-Cy7 (all from eBioscience, San Diego, CA) by flow cytometry (CytoFLEX XL, Beckman Coulter GmbH, Krefeld, Germany) as described previously [21 (link)] and HUVECs were isolated from the umbilical vein and characterized as published previously [24 (link)].
8
Characterizing Adipose-Derived Stem Cells
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The expression of cellular markers, including a hematopoietic marker (CD34) and mesenchymal markers (CD105, CD73, and CD90), in ADSCs (passage 3) was examined using flow cytometry (CD90-FITC, CD73-APC, CD105-PECy7, and CD34-PE antibodies from eBioscience were used, San Diego, CA).
Moreover, the ability of ADSCs in differentiating into osteoblasts and adipocytes was assessed. ADSCs cultured in 6-well plates grew to approximately 50% confluence. Then, the ADSCs were cultured in osteogenic differentiation medium or adipogenic differentiation medium (Gibco, Carlsbad, CA) for 21 days. ADSCs were stained with alizarin red S and oil red O to verify the osteoblast and adipocyte differentiation of ADSCs, respectively.
Moreover, the ability of ADSCs in differentiating into osteoblasts and adipocytes was assessed. ADSCs cultured in 6-well plates grew to approximately 50% confluence. Then, the ADSCs were cultured in osteogenic differentiation medium or adipogenic differentiation medium (Gibco, Carlsbad, CA) for 21 days. ADSCs were stained with alizarin red S and oil red O to verify the osteoblast and adipocyte differentiation of ADSCs, respectively.
9
Phenotypic Analysis of Adipose-Derived Stem Cells
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For ASC phenotype analysis, cells were detached with non-enzymatic cell dissociation solution (ATCC Manassas, VA, USA). In the next step, ASCs were washed with FACS buffer (phosphate-buffered saline, 0.1% NaN3, 1% FCS). Then, 5 × 104 cells were suspended in 50 μL of FACS buffer and stained with antibodies against the following surface markers: CD90-FITC, CD105-PE, CD73-APC (eBioscience, San Diego, CA, USA), CD34-PE-Cy7, CD45-PE, CD19-PE, and CD14-APC (BD Pharmingen, San Diego, CA, USA). After the washing step, cells were acquired and analysed using a FACSCanto cell sorter/cytometer and Diva software. The gating strategy is shown in Figure S1 in the Supplementary Materials .
10
Phenotyping of Mesenchymal Stem Cells
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In order to identify the mesenchymal stem cell phenotype, approximately 5 × 105 PDLSCs were incubated in 5% bovine serum albumin (BSA)/phosphate buffered saline (PBS) (Gibco, Invitrogen, Carlsbad, USA) 1 × at 4 °C in dark for 1 h with the following monoclonal antibodies: PAR1-FITC (Abcam, Cambridge, UK), OCT4-FITC (Abcam, Cambridge, UK), SOX2-FITC (Abcam, Cambridge, UK), STRO-1-FITC (Abcam, Cambridge, UK), CD14-FITC (eBioscience, San Diego, USA), CD90-FITC (eBioscience, San Diego, USA), CD31-PE (eBioscience, San Diego, USA), CD-44-PE (eBioscience, San Diego, USA), CD34-FITC (Biolegend, San Diego, USA) and CD146-PE (Biolegend, San Diego, USA) for 30 min at 4 °C. Unstained control was used to set gates. A total of 10–50,000 events were recorded and data analyzed through FlowJo (Becton Dickinson, Oregon, USA).
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