AD-MSC lines were washed with PBS and stained with ALP-APC (R&D) (1/50) and CD73-FITC (Biolegend) (1/160) for 25 min at 4 °C. Upon washing, the cell fractions (controls sorted, ALP+/CD73+, ALP−/CD73high, ALP−/CD73low) were sorted with a FACS BD Aria III 5L and seeded in Nunc™ 96-well plates (TPP) at a density of 1.2 × 104 cells/cm2 for osteogenic differentiation. Controls sorted were unstained cells processed through the FACS and collected without sorting specific subpopulations. Differentiation was induced 24 h after seeding. Freshly isolated SVFs were washed with PBS and stained with ALP-APC (R&D) (1/50), CD73-FITC (Biolegend) (1/160), and CD45-PE (Biolegend) (1/160) for 25 min at 4 °C. SVF fractions (controls sorted, CD45−/ALP+/CD73+, CD45−/ALP−/CD73high, CD45−/ALP−/CD73low) were sorted with FACS BD Aria III 5L and plated in vitro at a density of 1 × 104 in 96-well plates (TPP) for osteogenic differentiation. All media were changed every 4 days.
Cd73 fitc
Manufactured by BioLegend
Sourced in United States
CD73-FITC is a fluorescently labeled antibody that targets the CD73 cell surface protein. CD73 is an enzyme involved in the regulation of extracellular adenosine levels.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 fitc, by BioLegend (9 mentions)
Cd105 fitc, by BioLegend (6 mentions)
Cd34 fitc, by BioLegend (5 mentions)
Cd90 fitc, by BioLegend (4 mentions)
Cd90 pe, by BioLegend (4 mentions)
Cd44 apc cy7, by BioLegend (3 mentions)
Cd105 pe, by BioLegend (3 mentions)
Cd34 pe, by BioLegend (3 mentions)
Fitc hla a b c antibody, by BioLegend (2 mentions)
Percp cy5.5 cd146, by BioLegend (2 mentions)
Pe human ng2 mcsp, by BioLegend (2 mentions)
Bv421 ki67, by BioLegend (2 mentions)
Fitc anti s100, by Abcam (2 mentions)
Hla abc, by BD (2 mentions)
Cd36 apc, by Miltenyi Biotec (2 mentions)
Cd146 pe, by Miltenyi Biotec (2 mentions)
Cd31 fitc, by Thermo Fisher Scientific (2 mentions)
Cd19 pe, by BioLegend (2 mentions)
Hla dr pe, by BioLegend (2 mentions)
Cd29 pe, by BioLegend (2 mentions)
18 protocols using cd73 fitc
1
Osteogenic Differentiation of MSC Subpopulations
2
Multi-Parametric Flow Cytometry and Molecular Analyses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For multi-parametric flow cytometry, immunofluorescence/immunohistochemistry and DEPArray analyses, primary antibodies were obtained from the following sources: FITC-CD45 (#304054; 1:200), FITC-CD34 (#343504; 1:200), FITC-CD105 (#323204; 1:200), FITC-CD90 (#328108; 1:200), FITC-CD73 (#344016; 1:200), FITC HLA-A/B/C antibody (#311404; 1:200), PerCP/Cy5.5-CD146 (#342014; 1:100), PE-Human NG2/MCSP (#FAB2585P, 1:100), BV421-Ki67 (#350506; 1:100), were obtained from Biolegend, Inc. APC-Cy7-CD44 (#103028, 1:100) BV510-CD24 (#311126, 1:100), PE-Pan-Cytokeratin (#5075, 1:100) were purchased from Cell Signaling Technology, Inc. Anti-Mel-A antibody (# AC12-0297-03; 1:200) was obtained from Abcore, and FITC-Anti-S100 (#ab76749; 1:50) was purchased from Abcam. For immunohistochemistry, anti-human, anti-Mel-A antibody (# ab51061; 1:100), HLA-ABC (#565292; 1:100) were obtained from BD Biosciences, Inc. (San Jose, CA, USA). Anti-mouse secondary IgG anti-human antibodies used for IHC staining were received from Santa Cruz Biotechnology, Inc. Antibodies for immunofluorescence staining were obtained from Cell Signaling Technology, Inc. (1:500 dilution of stock solution), as previously described [3 (link),22 (link)]. NRF2 inhibitor ML385 (#2114) was obtained from Cayman, Inc. while Nodal inhibitor Lefty (746-LF/CF) was purchased from R&D Systems, Inc. (Waltham, MA, USA).
3
Multiparametric Flow Cytometry and Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For multiparametric flow cytometry, conjugated antibodies FITC‐CD45 (#304054; 1 : 200), FITC‐CD34 (#343504; 1 : 200), FITC‐CD105 (#323204; 1 : 200), FITC‐CD90 (#328108; 1 : 200), FITC‐CD73 (#344016; 1 : 200), FITC HLA‐A/B/C (#311404; 1 : 100), APC‐Cy7‐CD44 (#103028; 1 : 100), BV650‐CD44 (#103049; 1 : 100), and AF647‐Pan‐CK (#628604; 1 : 100) were obtained from BioLegend (San Diego, CA, USA); PE‐Pan‐CK (#5075; 1 : 100) antibody was received from Cell Signaling Technology (Danvers, MA, USA).
For immunohistochemistry, primary antibodies were obtained from the following sources: Anti‐human cocktail of gross cystic disease fluid protein‐15 (GCDFP‐15) + mammaglobin (#906H‐08; 1 : 200) was obtained from Sigma‐Aldrich (St. Louis, MO, USA); CD44 (#960‐MSM2‐P0; 1 : 200) and Pan‐CK (#MSM2‐371‐P0; 1 : 200) antibodies from NeoBiotechnologies (Union City, CA, USA); and Alexa Fluor‐conjugated anti‐mouse, anti‐rabbit secondary IgG antibodies (1 : 500) from Cell Signaling Technology.
For immunohistochemistry, primary antibodies were obtained from the following sources: Anti‐human cocktail of gross cystic disease fluid protein‐15 (GCDFP‐15) + mammaglobin (#906H‐08; 1 : 200) was obtained from Sigma‐Aldrich (St. Louis, MO, USA); CD44 (#960‐MSM2‐P0; 1 : 200) and Pan‐CK (#MSM2‐371‐P0; 1 : 200) antibodies from NeoBiotechnologies (Union City, CA, USA); and Alexa Fluor‐conjugated anti‐mouse, anti‐rabbit secondary IgG antibodies (1 : 500) from Cell Signaling Technology.
4
Multiparametric Flow Cytometry and Immunohistochemistry
5
Characterizing MenSCs and Their Mitochondria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MenSCs or MenSC-derived mitochondria were stained and labeled with anti-human fluorophore-conjugated antibodies for CD73-FITC (BioLegend, Inc.; Cat# 344015), CD90-FITC(BioLegend, Inc. Cat#328107), CD34-FITC (BioLegend, Inc. Cat# 343503), CD45-FITC (BioLegend, Inc. Cat# 304005), CD63-PE (BioLegend, Inc. Cat# 353003) and CD29-FITC (eBioscience; Thermo Fisher Scientific, Inc. Cat# 11029942) and detected by flow cytometric analysis. Briefly, trypsinized cells or mitochondria were washed then re-suspended in ice-cold PBS containing 1% BSA (Invitrogen; Thermo Fisher Scientific, Inc.). Fluorophore-conjugated antibodies were added at concentrations recommended by the manufacturer's protocols (95 µl staining buffer, 5 µl fluorophore-conjugated antibodies) and incubated in the dark for 30 min, 25˚C. The cells or mitochondria were washed twice in staining buffer (eBioscience, Thermo Fisher Scientific, Inc.) and analyzed under a flow cytometer (LSR Fortessa; BD Biosciences). Data analysis was performed using FlowJo (BD Biosciences. v.7.6.5).
6
Flow Cytometry Characterization of hASCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each assay contained a unique combination of the following mouse anti-human monoclonal antibodies: CD73-FITC, CD90-APC, CD105-PE (BioLegend, San Diego, CA, USA), CD34-PE, CD36-APC, CD146-PE (Miltenyi BioTech, Bergisch, Germany), CD61-PE (Beckman Coulter Inc., Pasadena, CA, USA), CD15-FITC, and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA). All antibodies were titrated and used at a 50 ng/assay concentration. Isotype controls and specific mAbs were employed at the same final concentrations. Routinely, 50,000 hASCs (in 100 µL FACS buffer) were mixed with the appropriated antibody combination and incubated for 15 min at RT in the dark. Finally, the sample was diluted with 100 µL FACS buffer before the acquisition, according to our flow cytometer procedure (see Section 2.3.3 ).
7
ECFC and MSC Immunophenotyping Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ECFC single-cell suspension was generated by detaching cells with TrypLE™ Express Enzyme (Gibco, USA) and resuspended to a concentration of 1 × 107 cells/ml. Samples were incubated, respectively, with anti-human CD31-FITC (eBioscience, USA), VEGFR2/KDR-PE (R&D, USA), CD144-FITC (Abcam, UK), CD34-PE (Biolegend, USA), CD45-FITC (Biolegend, USA), and CD14-FITC (eBioscience, USA). MSCs were resuspended to a concentration of 1 × 106 cells/ml and incubated, respectively, with anti-human CD29-PE (Biolegend, USA), CD90-PE (Biolegend, USA), CD14-FITC (Biolegend, USA), CD19-PE (Biolegend, USA), CD73-FITC (Biolegend, USA), CD105-FITC (Biolegend, USA), HLA-DR-PE (Biolegend, USA), CD34-PE (Biolegend, USA), CD45-FITC (Biolegend, USA), and CD31-FITC (eBiosciences, USA). 5 μl antibody solution was added into 100 μl cell suspension and incubated for 30 minutes at 4°C in the dark; 400 μl of PBS was added and cells were analyzed with FACSAria I (Becton Dickinson, USA) or Accuri C6 (Becton Dickinson, USA) and Becton Dickinson CELLQuest software.
8
Expansion and characterization of human BMD-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The AGS cell line (human gastric adenocarcinoma cell line) was purchased from Iran Pasteur Institute (Tehran, Iran) and were cultured in RPMI 1640 supplemented with 10% FBS (Gibco, Manchester, UK) at 37°C in a humid incubator with 5% CO2. Human BMD-MSCs were expanded in vitro from the bone marrow of healthy donors after informed consent was obtained. Mononuclear cells (MNCs) were isolated by gradient centrifugation at 2,500 rpm for 30 min on Ficoll-Paque™ Plus (Amersham Pharmacia Biotech, Uppsala, Sweden). Then, plated at a concentration of 20-30 × 106 cells/cm2 in Dulbecco's Modified Eagle Medium (DMEM) containing 20% (v/v) of FBS. Then, nonadherent cells were removed 2 days later and a fresh medium was added. BMD-MSCs were trypsinized when the cultures reached 80-100% confluence and subcultured. The purity of MSC suspensions was assessed by flow cytometry using the following monoclonal antibodies: anti-CD34-FITC, CD45-FITC, CD73-FITC (all from Biolegend, Ankara, Turkey), and Stro-1 (R&D, Istanbul, Turkey).
9
Characterization of Human Olfactory Mesenchymal Stem Cells by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated hO-MSCs were characterized using fluorescence-activated cell sorting (FACS). At least 100,000 isolated hO-MSCs were used for the analysis of each cell surface marker. Briefly, hO-MSCs were harvested and stained with the cell surface marker antibodies described below for 30 min at 4 °C in the dark and analyzed using a flow cytometer (FACS Canto II, Becton Dickinson and Company, Franklin Lakes, NJ, USA) at the Soonchunhyang Biomedical Research Core Facility of the Korea Basic Science Institute (KBSI). Antibodies were diluted 1:20 with FACS buffer containing 1% BSA in PBS and fluorescence-labeled cells were kept on ice during FACS analysis. MSC-positive markers CD29-PE (cat# 303,004, BioLegend, San Diego, CA, USA), CD44-FITC (cat# 103,022, BioLegend, San Diego, CA, USA), CD73-FITC (cat# 344,016, BioLegend, San Diego, CA, USA), CD105-PE (cat# 12–1057-42, eBioscience, San Diego, CA, USA), and CD166-PE (cat# 343,904, BioLegend, San Diego, CA, USA); and MSC-negative markers CD45-FITC (cat# 368,508, BioLegend, San Diego, CA, USA), CD34-FITC (cat# 343,517, BioLegend, San Diego, CA, USA), and CD31-PE (cat# 303,106, BioLegend, San Diego, CA, USA) were used for flow cytometry.
10
Optimizing Mesenchymal Stem Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Collagenase type I, penicillin/streptomycin (Pen/Strep) broad-spectrum antibiotic cocktail, trypsin-EDTA (0.25%), fetal bovine serum (FBS), human insulin and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). VersaLyseTM was purchased from Beckman Coulter (Miami, FL, USA). Dexamethasone, 3-isobutyl-methylxanthine, Nile Red (NR) and indomethacin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vybrant® DyeCycleTM Violet was purchased from Thermo Fisher Scientific/Life Technologies (Eugene, OR, USA). The following mouse anti-human monoclonal antibodies were purchased from Biolegend (San Diego, CA, USA): CD14-APC Cy7 (Clone M5E2), CD31-PE Cy7 (Clone WM-59), CD36-APC (Clone 5-271), CD73-FITC (Clone AD2), CD44-APC Cy7 (Clone IM7) and CD105-PE (Clone 42A3). Mouse anti-human CD45-Krome Orange (Clone J.33), CD90-PE-Cy5 (Clone Thy-1), CD34-PE Cy7 (Clone 581), and the viability dye, 7-aminoactinomycin D (7-AAD) were purchased from Immunotech/Beckman Coulter (Marseille, France).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!