Surface markers for hADSCs were evaluated using a mini Guava EasyCyte flow cytometer. 5x104 cells were incubated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies, anti-CD105, anti-CD34, anti-STROI, anti-CD73, anti-CD45, anti-HLA-ABC, anti-HLA-DR (Invitrogen, Frederick, MD) for 30 min at 4°C in PBS and washed afterwards. Cell autofluorescence in channel F1 or F2 was subtracted to obtain a neat signal of each marker. 5x104 cells were plated for differentiation tests. Adipogenic differentiation was performed in StemPro adipogenesis-conditioned medium (Invitrogen, Grand Island, NY) for one week, replacing medium every 3 days. Lipid droplets were stained by using red oil staining for validating differentiation. StemPro osteogenesis-conditioned medium (Invitrogen, Grand Island, NY) was used for osteogenic differentiation for 11 days, replacing media every 48 hours. Extracellular calcium deposits were identified by Von Kossa staining. hADSCs cultured in DMEN were also stained as controls.
Anti cd73
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD73 is a laboratory reagent used for the detection and analysis of the CD73 protein. CD73 is an enzyme involved in the extracellular metabolism of adenosine monophosphate (AMP) to adenosine. This reagent can be used in various applications, such as flow cytometry, to identify and quantify cells expressing CD73.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd105, by Thermo Fisher Scientific (6 mentions)
Anti cd45, by Thermo Fisher Scientific (3 mentions)
Anti cd34, by Thermo Fisher Scientific (2 mentions)
Anti cd90, by Thermo Fisher Scientific (2 mentions)
Anti cd29, by Thermo Fisher Scientific (2 mentions)
Anti hla dr, by Thermo Fisher Scientific (1 mentions)
Anti hla abc, by Thermo Fisher Scientific (1 mentions)
Anti f4 80, by Thermo Fisher Scientific (1 mentions)
Nestin, by Thermo Fisher Scientific (1 mentions)
Anti cd31, by Thermo Fisher Scientific (1 mentions)
Anti epcam, by Abcam (1 mentions)
Anti cd26, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg pe cy5, by Thermo Fisher Scientific (1 mentions)
Anti sca1, by BD (1 mentions)
Anti cd44, by BD (1 mentions)
Anti cd117, by BD (1 mentions)
Anti cd11b, by BD (1 mentions)
Anti cd90, by BD (1 mentions)
Anti cd45, by BD (1 mentions)
Anti cd31, by BD (1 mentions)
7 protocols using anti cd73
1
Characterization and Differentiation of hADSCs
2
Isolation and Characterization of PDLSCs
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Fourth-generation PDLSCs were collected and washed twice with PBS containing 1% FBS. After incubation in the dark with anti-CD45, anti-CD31, anti-CD29, anti-CD90 and anti-CD105 antibodies (Invitrogen, California, USA) at 4 °C for 30 minutes. The cells were centrifuged and washed three times, and the suspension was analyzed by sorted flow cytometry (Influx, BD, New Jersey, USA). The detection of DPSC surface antibodies was the same as above, and the markers included anti-CD73, anti-CD90, anti-CD45, anti-CD31 and Nestin (Invitrogen, California, USA).
Macrophage polarization markers were detected as follows. After washing with PBS, centrifuged cells with anti-F4/80 and anti-CD86 or anti-CD206 (Invitrogen, California, USA) were incubated in the dark at 4 °C for 30 minutes. The cells were then washed once, suspended in PBS containing 1% FBS and analyzed by flow cytometry. Flow cytometry data were analyzed by FlowJo software v10.2 (BD, New Jersey, USA).
Macrophage polarization markers were detected as follows. After washing with PBS, centrifuged cells with anti-F4/80 and anti-CD86 or anti-CD206 (Invitrogen, California, USA) were incubated in the dark at 4 °C for 30 minutes. The cells were then washed once, suspended in PBS containing 1% FBS and analyzed by flow cytometry. Flow cytometry data were analyzed by FlowJo software v10.2 (BD, New Jersey, USA).
3
Multimarker Flow Cytometry Analysis
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For flow cytometry analysis, 2 × 105 cells were washed with phosphate-buffered saline (PBS) and then stained with the following antibodies: anti-CD11b (BD Pharmingen, Franklin Lakes, NJ, USA), anti-CD29 (eBioscience, San Diego, CA, USA), anti-CD31 (BD Pharmingen), anti-CD34 (BD Pharmingen), anti-CD44 (BD Pharmingen), anti-CD45 (BD Pharmingen), anti-CD73 (eBioscience), anti-CD105 (eBioscience), anti-CD90 (BD Pharmingen), anti-CD117 (BD Pharmingen), anti-Sca1 (BD Pharmingen), anti-CD26 (eBioscience), anti-EpCAM (Abcam), and goat anti-rabbit IgG PE-Cy5.5 (Thermo Fisher Scientific).
4
Mitochondrial and Immune Profiling of MSCs
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Mitochondria in MSCs cultured either with CiMS or Cellartis were stained with AIE Mitochondria Red (AIEgen Biotech Co.) and observed by confocal microscopy (Olympus; FV1200) using a 40× objective lens. Mitochondrial membrane voltage and cell size were evaluated by flow cytometry (BD Aria II) after staining with AIE Mitochondria Red (AIEgen Biotech Co.). Surface antigens of MSCs were detected by flow cytometry after staining with anti-CD73 (eBioscience; cat. no. 17-0739-42), anti-CD90 (BioLegend; cat. no. 328121), and anti-CD105 (eBioscience; cat. no. 12-1057-42) antibodies in accordance with the supplier’s instructions. Tregs were detected by flow cytometry after harvesting PMBCs and staining with anti-CD3 (BD Biosciences; cat. no. 563798), anti-CD4 (BD Biosciences; cat. no. 561841), anti-CD8 (BD Biosciences; cat. no. 557750), anti-CD25 (BD Biosciences; cat. no. 560990), and anti-FOXP3 (BD Biosciences; cat. no. 560852) antibodies.
5
Immunophenotypic Analysis of Single Cells
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Single cells were collected with 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then resuspended in cold PBS at a concentration of 5×106 cells/mL. Then, the cells were incubated in the dark on ice for 30 min using antibodies including anti-CD29, anti-CD34, anti-CD45, anti-CD73, anti-CD90, anti-CD105 and anti-CD146. All antibodies were purchased from BioLegend, Inc. Subsequently, cells were twice washed and 500 μL PBS resuspension for each sample. The study was performed by Becton-Dickinson Accuri C6 (BD Biosciences, San Jose, CA, USA). FlowJo (version 10.0.7 r2) was used for the data analysis.
6
Phenotypic Characterization of Cultured MSCs
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Cultured MSCs at passage 5 were detached using 0.25% trypsin/EDTA and centrifuged at 400 × g for 5 min. The supernatant was discarded and the cell pellet was re-suspended in D-PBS containing 1% fetal bovine serum to the concentration of 105 cells/ml. Cells were then incubated with 10 µl of phycoerythrin-conjugated antibodies anti-CD34 (1:50, cat. no. MA1-19645; Thermo Fisher Scientific, Inc.), anti-CD44 (1:50, cat. no. MHCD4404; Thermo Fisher Scientific, Inc.), anti-CD73 (1:50, cat. no. FAB5795P; R&D Systems, Inc., Minneapolis, MN, USA), anti-CD90 (1:50, cat. no. A15794; Thermo Fisher Scientific, Inc.), anti-CD105 (1:50, cat. no. MA1-80944; Thermo Fisher Scientific, Inc.) at 25°C for 45 min. Then cells centrifuged at 300 × g for 15 min and washed with flow cytometry washing buffer (PBS with 0.5–1% bovine serum albumin; Sigma-Aldrich; Merck Millipore), the cells analyzed by flow cytometry analyzer (BD FACSARIA III, BD Biosciences).
7
Murine Bone Marrow Cell Immunophenotyping
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As previously described [29 (link)], bone marrow cells of femur and tibia were harvested and pooled together. After red blood cells were lysed, the bone marrow cells were centrifuged and then the cell pellet was resuspended and fixed in 4% paraformaldehyde. Cells were permeabilized in 0.1% Triton X-100 and then blocked in blocking buffer (PBS with 3% FBS and 0.1% sodium azide) for 30 min on ice. Then the cells were incubated with anti-CD73 (12–0731-83, ThermoFisher scientific), anti-Sca1 (11–5981-85, ThermoFisher scientific) or isotype control for 1 h at 37 °C in dark room, and then washed twice with PBS with 0.1% BSA. Probes were analyzed using a BD Calibur flow cytometer and CellQuest software (Becton Dickinson).
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