Cultured synovial MSCs from three donors at passage 1 were harvested using a cell-dissociation buffer. Cells were suspended in HBSS at a density of 5 × 105 cells/mL and stained for 30 minutes on ice with the antibodies CD31-PE-Cy7 (Becton, Dickinson and Company; BD, Franklin Lakes, NJ, USA), CD45-APC-H7 (Biolegend, San Diego, CA, USA), CD44-APC-H7 (BD), CD73-BV421 (BD), CD90-PE (BD), CD105-PerCP-Cy5.5 (BD), CD140a-BV421 (BD), CD140b-PerCP-Cy5.5 (BD), CD146-FITC (BD) and CD271-APC (Miltenyi Biotec) for cell surface analysis. Flow cytometric analysis of the cell surface was performed by a triple-laser FACS Verse™ system (BD).
Cd44 apc h7
Manufactured by BD
Sourced in United States
CD44-APC-H7 is a fluorescently-labeled antibody that binds to the CD44 surface protein. CD44 is a cell adhesion molecule involved in various cellular processes. The APC-H7 fluorophore is attached to the antibody, allowing for detection and analysis of CD44-expressing cells using flow cytometry.
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Lab products found in correlation
Cd90 pe, by BD (4 mentions)
Facsverse system, by BD (4 mentions)
Cd105 percp cy5, by BD (4 mentions)
Cd73 bv421, by BD (3 mentions)
Cd45 fitc, by BD (3 mentions)
Cd31 fitc, by BD (3 mentions)
Cd31 pe cy7, by BD (2 mentions)
Cd140b percp cy5, by BD (2 mentions)
Cd271 apc, by Miltenyi Biotec (2 mentions)
Cd146 fitc, by BD (2 mentions)
Cd11b pe, by BD (2 mentions)
Cd14 apc, by BD (2 mentions)
Cd206 fitc, by BD (2 mentions)
Cd45 apc h7, by BioLegend (1 mentions)
Cd140a bv421, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Cd90 fitc, by BD (1 mentions)
Respective isotype controls, by BD (1 mentions)
Cd73 pe cy7, by BD (1 mentions)
Cd45 pe cy7, by BD (1 mentions)
7 protocols using cd44 apc h7
1
Characterizing Synovial MSC Surface Markers
2
Flow cytometry-based hMSC subpopulation enrichment
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For flow cytometry, fixed cells were immunostained using antibodies CD44-APC-H7, CD73-PE-CY7, CD90-FITC, CD105-PerCP-Cy5.5, CD146-V450, and respective isotype controls (BD Bioscience) and analyzed using a flow cytometer (LSR II, BD). To enrich for α-SMA-positive and α-SMA-negative hMSC populations, cells were trypsinized and sorted for cell size (upper and lower 25% in forward scatter) using fluorescence activated cell sorting (FACS) (FACSAria III, BD).
3
Synovial Cell Isolation and Characterization
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Synovium was digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C. After 3 h, the digested cells were filtered through a 70-μm cell strainer (Greiner Bio-One GmbH, Kremsmunster, Austria). The cells from six donors were harvested using a cell-dissociation buffer. Cells were suspended in HBSS at a density of 5 × 105 cells/mL and stained for 30 min on ice with the antibodies. For cell isolation, cells were stained with CD31-PE-Cy7 (BD), CD45-PE-Cy7 (BD), CD235a-PE-Cy7 (BD), CD55-FITC (Miltenyi Biotec), CD90-PE (BD) and CD271-APC (Miltenyi Biotec) were used at day 0. Flow cytometric isolation of cell surface antigens were performed by a double-laser Aria 2 system (BD). For cell surface analysis, cells were stained with CD31-FITC (BD), CD45-FITC (BD), CD44-APC-H7 (BD), CD73-BV421 (BD), CD90-PE (BD), CD105-PerCP-Cy5.5 (BD), CD55-PE (BD), CD271-APC (Miltenyi Biotec), CD140b-PerCP-Cy5.5 (BD) and CD146-FITC (BD) at passage 3. Flow cytometric analysis of cell surface antigens was performed by a triple-laser FACS Verse system (BD). These data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Flow cytometric analyses were also performed for expanded cells at passage 3.
4
Multiparametric Immune Cell Profiling
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Passage 2 cells were suspended in Hank’s Balanced Salt Solution (HBSS) at a density of 5 × 105 cells/mL and stained for 30 min on ice with the following antibodies: CD11b-PE, CD11c-PE-Cy7, CD14-APC, CD31-FITC, CD44-APCH7, CD45-FITC, CD73-BV421, CD90-PE, CD105-PerCP-Cy5.5, CD206-FITC, and HLADR-APC (BD, Franklin Lakes, NJ). Cell surface antigens were analyzed using a triple-laser FACS Verse™ system (BD).
5
Characterization of Human Synovial MSCs
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Human synovial MSCs were detached with trypsin and suspended in PBS containing 2% FBS and 5 mM EDTA. The MSCs were then stained for 30 min at 4 °C with CD44-APCH7, CD73-V450, CD90-PE-Cy7, CD105-APC, CD34-PE, and CD45-PerCP-Cy5.5 antibodies (1:200; BD Biosciences), using Ghost Dye Violet (1:1000; Tonbo Biosciences, San Diego, CA, USA) to remove dead cells. Isotype controls were prepared as negative controls. The percentage of antigen-positive cells was evaluated using a FACSVerse II system (BD Biosciences).
6
Phenotypic Analysis of RA and OA Synovium Cells
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Flow cytometric analyses of synovium-derived cells from RA and OA were performed with digested cells before plating (day 0) and expanded cells cultured for 14 days. The cells from three donors were harvested using a cell dissociation buffer. Cells were suspended in HBSS at a density of 5 × 105 cells/mL and stained for 30 minutes on ice with the antibodies CD11b-PE, CD11c-PE-Cy7, CD14-APC, CD31-FITC, CD44-APC-H7, CD45-FITC, CD73-BV421, CD90-PE, CD105-PerCP-Cy5.5, CD206-FITC, and HLA-DR-APC (Becton, Dickinson and Company; BD, Franklin Lakes, NJ, USA). Flow cytometric analysis of cell surface antigens was performed by a triple-laser FACS Verse™ system (BD).
7
Comprehensive Breast Cancer Cell Profiling
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Cells were prepared from three separate cultures as previously described [14] . Single cell suspensions were stained with SYTOX BLUE (Molecular Probes), CD49f-PE-Cy5, EpCAM-FITC, CD44-APC H7 and CD24-PE (Becton-Dickinson (BD); discrimination was also performed by gating out cells with disproportionate forward scatter height and area. Positivity thresholds were determined based on the signal from isotype controls for each marker and cell line combination (Fig- S1). At least 1x10 4 events representing live, single cells were collected for each sample. Fluorescence compensation was performed on each occasion, then retrospectively checked and modified if necessary using FCS Express software (v6.0; DeNovo). Population frequencies were determined for individual and combined parameters from an average of three cultures (consecutive passages).
The proportions of luminal-1, luminal-2, basal and mesenchymal subpopulations were determined as described [2] .
The proportions of luminal-1, luminal-2, basal and mesenchymal subpopulations were determined as described [2] .
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