FACS sorted CD49f- cells or cultured CD271+CD49f- cells were stained with primary antibodies PE-conjugated CD271, PE-conjugated PDGFRB (1.25 μg/ml, mouse IgG1; R&D Systems), PE-conjugated SUSD2, (W5C5 clone, 1:20, mouse IgG1; Biolegend), APC-conjugated CD90 (25 μg/ml, mouse IgG1κ, BD Pharmingen), CD146 (1:2, supernatant, clone CC9, mouse IgG2a; donated by Prof David Haylock, CSIRO, Clayton, Victoria, Australia), CD73 (10 μg/ml, mouse IgG1κ; BD Pharmingen) or CD105 (10 μg/ml, mouse IgG1ƙ; BD Pharmingen). The CD146 samples were incubated with secondary antibody fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG2a (5 μg/ml, clone R19-15; BD Pharmingen). CD73 and CD105 samples were incubated with secondary antibody PE-anti-mouse IgG1 (2 μg/ml, clone A85-1, BD Pharmingen). Isotype matched controls or unlabelled controls were included for each antibody and used to set the electronic gates on the flow cytometer. Cells were then incubated with Sytox Blue and analysed by FACS Canto II analyser (BD Biosciences). FACS data were analysed by FACSDiva software (BD Biosciences).
Apc conjugated cd90
Manufactured by BD
Sourced in United States
APC-conjugated CD90 is a laboratory reagent used in flow cytometry analysis. It is a monoclonal antibody conjugated with the fluorescent dye Allophycocyanin (APC) that binds to the CD90 (Thy-1) cell surface antigen. CD90 is expressed on various cell types, including hematopoietic stem cells, mesenchymal stem cells, and certain T cell subsets. This reagent can be used to identify and enumerate CD90-positive cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Apc conjugated susd2, by BioLegend (2 mentions)
Pe conjugated cd140b, by R&D Systems (2 mentions)
Facscanto 2, by BD (2 mentions)
Anti mouse igg1 pe, by BD (1 mentions)
Facscanto 2 analyser, by BD (1 mentions)
Fitc conjugatedanti mouse igg2a, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Mouse igg1, by BD (1 mentions)
Cd105, by BD (1 mentions)
Cd146, by BD (1 mentions)
Pe conjugated cd44, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated cd45, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Pe conjugated cd31, by BioLegend (1 mentions)
Pe conjugated cd29, by BioLegend (1 mentions)
Pe cy7 conjugated cd45, by BioLegend (1 mentions)
5 protocols using apc conjugated cd90
1
Immunophenotyping of CD49f- and CD271+CD49f- Cells
2
Phenotypic Characterization of eMSCs
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The surface phenotype of untreated and A83-01-treated eMSC was assessed by flow cytometry for three MSC markers [SUSD2, CD140b (Platelet-derived growth factor receptor β) and CD90 (Thy-1)] as described previously (Gurung et al., 2015 ). Cells were incubated for 1 h with 1:20 primary or matched isotype control antibodies in PBS containing 2% FCS at 4°C in dark. The primary antibodies were APC-conjugated SUSD2 (Biolegend), PE-conjugated CD140b (R&D Systems, United States) and APC-conjugated CD90 (BD Pharmingen, United States). Cells were washed with 2% FCS/PBS and fixed with 1:1 4% paraformaldehyde in 2% FCS/PBS. eMSC were analyzed using BD FACSCanto II (BD) (10,000 events/sample) and FlowJo v.10 software.
3
Evaluating eMSC Surface Phenotype
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The surface phenotype of untreated and A83-01-treated eMSC was assessed by flow cytometry for three MSC markers [SUSD2, CD140b (Platelet-derived growth factor receptor β) and CD90 (Thy-1)] as described previously. 23 Cells were incubated for 1 h with 1:20 primary or matched isotype control antibodies in PBS containing 2% FCS at 4°C in dark. The primary antibodies were APC-conjugated SUSD2 (Biolegend), PE-conjugated CD140b (R&D Systems, USA) and APC-conjugated CD90 (BD Pharmingen, USA). Cells were washed with PBS/2% FCS and fixed with 4% paraformaldehyde in PBS/2% FCS. eMSC were analysed using BD FACSCanto II (BD) (10,000 events /sample) and FlowJo v.10 software.
4
Immunophenotyping of Passage-1 Cells
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Passage-1 cells (1 × 106 cells) were suspended in 100 μl phosphate buffered saline (PBS) containing 10 μg/ml antibodies for APC-conjugated CD90 (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated CD44 (eBioscience, San Diego, CA, USA), FITC-conjugated CD45 (eBioscience), and PerCP-Cyanine5.5-conjugated CD11b (Biolegend, San Diego, CA, USA). As an isotype control, nonspecific anti-mouse IgG coupled with APC, PE, FITC, or PerCP-Cyanine5.5 (Becton Dickinson) was substituted for the primary antibody. After incubation for 30 minutes at 4°C, the cells were washed with PBS and then resuspended in 500 μl of PBS for analysis. Cell fluorescence was evaluated by flow cytometry using a FACSVerse instrument (Becton Dickinson); the data were analyzed using Flowjo software (Tree Star, Ashland, OR, USA). All analyses were performed on samples from all 6 donors. The data were analyzed with the Kruskal Wallis test, followed by Dunn’s multiple comparisons test.
5
Isolation and Characterization of GFP-Labeled Mesenchymal Stem Cells from Synovium
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1 Â 10 6 GFP þ MSCs were injected 1week after the ACLT, then synovium was harvested 1day after the injection, following digestion with collagenase V for three hours. After filtering through a 70 mm cell strainer and centrifuging at 1500 rpm for 5 min, cells were suspended in ice-cold Hank's balances salt solution and then stained for 30 min on ice with a monoclonal antibody of APCconjugated CD90 (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Synovial fluid was also extracted from the same knee, and prepared in the same manner without collagenase digestion. Propidium iodide (PI) fluorescence was measured, and a live cell gate was defined that excluded the cells positive for PI. Additional gates were defined as positive for GFP and CD90. Double positive cells were further analyzed for PE-conjugated CD29, PEconjugated CD31, and PEcy7-conjugated CD45 (Biolegend, San Diego, CA, USA). Flow-cytometric analysis and sorting were performed on a MoFlo (Beckman Coulter, Brea, CA, USA), and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA) 26 .
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