Bone marrow MNCs were defrosted and the MSC frequency was measured based on the CD45-CD271+ phenotype, as previously described [24 (link)], with some modifications. Bone marrow MNCs were re-suspended at 1 x 107 cells/ml in FACs buffer (PBS +0.5% bovine serum albumin (BSA) +2 mM EDTA). Antibodies were added at the manufacturers’ recommended concentrations and the cells were incubated with the antibodies for 20 minutes. Antibody combinations used were: CD45-PeCy7/CD271-APC/CD140a-PE, CD45-PeCy7/CD271-APC/CD140b-PE or an isotype controls combination (CD271-APC was from Miltenyi Biotec, Bergisch Gladbach, Germany, all other antibodies from BD Biosciences, Oxford, UK). The cells were washed and re-suspended in FACs buffer containing 100 ng/ml 4',6-diamidino-2-phenylindole (DAPI) before analysing on a BD LSRII flow cytometer. Dead cells were excluded from the analysis using DAPI before gating on the CD45-CD271+ population and assessing the expression of CD140a and CD140b (PDGF receptors α and β, respectively) on these cells.
Cd271 apc
Manufactured by Miltenyi Biotec
Sourced in Germany, United Kingdom
CD271-APC is a fluorochrome-conjugated antibody specific for the CD271 (p75 NGFR) cell surface marker. CD271 is expressed on various cell types, including nerve cells and their progenitors. The APC (allophycocyanin) fluorochrome allows for detection of CD271-positive cells using flow cytometry or other fluorescence-based applications.
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Lab products found in correlation
Cd90 pe, by BD (3 mentions)
Cd45 percp, by Miltenyi Biotec (3 mentions)
Cd45 fitc, by BD (2 mentions)
Cd31 pe cy7, by BD (2 mentions)
Cd45 pe cy7, by BD (2 mentions)
Facsverse system, by BD (2 mentions)
Cd105 percp cy5, by BD (2 mentions)
Cd44 apc h7, by BD (2 mentions)
Cd73 bv421, by BD (2 mentions)
Cd140b percp cy5, by BD (2 mentions)
Cd146 fitc, by BD (2 mentions)
Igg1 pe, by Miltenyi Biotec (2 mentions)
Cd73 fitc, by Thermo Fisher Scientific (2 mentions)
Igg1 apc, by Miltenyi Biotec (2 mentions)
Igg2a percp, by Miltenyi Biotec (2 mentions)
Human cd105 pe, by Thermo Fisher Scientific (2 mentions)
Cd44 fitc, by Miltenyi Biotec (2 mentions)
Cd73 pe, by BD (2 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Cd45 pe cy7, by Miltenyi Biotec (1 mentions)
10 protocols using cd271 apc
1
Quantification of Bone Marrow MSCs
2
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.
3
Characterizing Synovial MSC Surface Markers
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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).
4
Characterization of ATD-MSCs by Flow Cytometry
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Cell surface markers of ATD-MSCs were analysed by flow cytometry on fresh SVF and on SVF derived after thawing of adipose tissue and its collagenase treatment. ATD-MSCs were identified as CD105, CD44, CD73, and CD271 positive cells and negative for CD45 expression. Standard labelling protocol was performed with the following antibodies fluorochrome-conjugated and isotypic controls: human CD105 PE (Invitrogen), CD73 FITC (kindly provided by Professor Malavasi, University of Turin), CD44 FITC, CD45 PerCP, CD3 PerCP, CD271 APC, IgG1 PE, IgG1 APC, and IgG2a PerCP (Miltenyi Biotec), and IgG1 FITC-conjugated (Immunostep). About 105 events/sample were used for capture with CellQuest software. All data were analysed with FlowJo software (Tree Star).
5
Surface Marker Analysis of Sorted Cells
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Cells from the sorting procedure were centrifuged at 300g for 10 min and pellets resuspended in the appropriate fluorescent antibodies or isotype control antibodies at a concentration of 1:11 in MACS buffer. Cells were incubated in up to three antibodies for 10 min at 4 °C. After a final wash step, cells were resuspended in MACS buffer and transported directly for flow cytometry and analysed for surface marker expression using a Cyan ADP flow cytometer (Beckman Coulter, High Wycombe, UK) at the Faculty of Biology, Medicine and Health Core Facility, University of Manchester. Compensation was carried out using a bead kit (Miltenyi Biotec, Surrey, UK, 130-097-900). All antibodies and isotype controls used were obtained from Miltenyi Biotech (Surrey, UK) and are as follows with product codes: CD29-PE (130-101-275), CD34-PE-Vio770 (130-100-844), CD45-PerCP (130-098-145), CD90-FITC (130-097-930), CD146-VioBlue (130-099-678), CD271-APC (130-091-884), Mouse IgG1-PE (130-098-845), Mouse IgG2a-PE-Vio770 (130-098-564), Mouse IgG2a-PerCP (130-099-190), Mouse IgG1-FITC (130-098-847), Mouse IgG1-VioBlue (130-099-756), and Mouse IgG1-APC (130-098-846). Data were analysed using FlowJo v10 (FlowJo LLC, Ashland, OR, USA).
6
Mesenchymal Cell Surface Marker Analysis
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Mesenchymal cell surface markers were analysed by flow cytometry on fresh SVF and on cultured ASCs. A standard labelling protocol was performed with the following antibody fluorochrome-conjugated and isotypic controls: human CD105 PE (Invitrogen), CD73 FITC (kindly provided by Prof. Malavasi, University of Turin), CD44 FITC, CD45 PerCP, CD271 APC, IgG1 PE, IgG1 APC and IgG2a PerCP (Miltenyi Biotec), and IgG1 FITC conjugate (IMMUNOSTEP). About 105 events/sample were used for capture with CellQuest software. All data were analysed with Flowlogic software (Miltenyi Biotec).
7
Flow Cytometry Phenotyping of Expanded Colonies
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Colonies expanded from blood were trypsinised, washed and stained with antibodies: CD31-FITC, CD90-PE, CD105-PE (AbD Serotec, Kidlington, UK), CD19-PE, CD33-FITC, CD34-PerCp, CD45-PE-Cy7, CD61-FITC, CD73-PE, (BD Biosciences, Oxford, UK) and CD271-APC (Miltenyi Biotec), at manufacturers recommended concentrations. Cells were washed, 30,000 events captured on a LSRII flow cytometer and the data analysed using FACSDiva Software (both BD Biosciences).
8
Western Blot Antibody Compendium
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The following antibodies used for western blot analyses were obtained from Cell Signaling Technology (unless otherwise indicated): phospho-AKTS473 (#9271), phospho-ERK1/2 (#4370), phospho-JAK1 (#3331), phospho-JAK2 (#3771), phospho-MEK1/2 (#9154), phospho-mTOR (#2971), phospho-p70S6Kinase (#9204), phospho-STAT1 (#9167), phospho-STAT3 (#9145), phospho-STAT5 (#9351), phospho-TYK2 (#9321), DYKDDDDK (#2368), CD127 (anti-IL7Ra; R&D Systems, Minneapolis, MN, USA; #MAB306), RAS (Merck Millipore; #05-516) and β-actin (Sigma-Aldrich; #2547). The following antibodies used for flow cytometry were obtained from Miltenyi Biotec: CD127-FITC (#130-094-888) and CD271-APC (#130-091-884).
9
Immunophenotyping of SVF Cells
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SVF cells were separated into CD45+ and CD45− cells using microbeads and autoMACS pro separator (Miltenyi) according to the manufacturer’s instructions. CD45− cells were incubated with Fc Block (BD Biosciences) prior to staining. Then, cells were labeled with following antibodies: human CD31-FITC (BD Biosciences, clone WM59, used in 1:20 dilution), CD271-APC (Miltenyi, cloneME20.4–1.H4, 1:20), and CD34-peridinin chlorophyll protein (PerCP) (BD Biosciences, clone 8G12, 1:20) for 15 min at 4°C. Then, cells were washed twice in PBS with 2.0% FBS. Flow cytometric data were acquired by using Accuri C6 (BD Biosciences). Before immunophenotyping, the instrument performance was validated by using BD Accuri Spherotech Validation Beads (BD Biosciences). A total of 30,000–100,000 events were acquired at slow flow rate. Acquired data were analyzed using FlowJo software X 10.0.7 (Tree Star Inc.). Isotype-matched negative controls or fluorescence-minus controls were used to define the threshold for each specific signal and establish the appropriate gate for positive cells.
10
Phenotypic Profiling of Mesenchymal Stem Cells
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The phenotypic profile of MSCs was routinely analysed at P0 and P2 using flow cytometry for expression of a panel of surface antigen markers: CD45 FITC, CD14 PE, CD44 FITC, CD166 PE, CD90 PE, CD73 PE, HLA-DP -DQ -DR FITC, HLA-ABC FITC (BD Pharmingen), CD105 PE (Ancell) and CD271 APC (Miltenyi Biotec S.r.l.). Nonspecific fluorescence and morphological cell parameters were determined by incubation of the same cell aliquot with isotype-matched unrelated mouse immunoglobulin (IgGs) (BD Pharmingen). Flow cytometry was performed by collecting 10 4 events on a FACS Calibur system equipped with a 488 nm argon laser (Becton Dickinson). Data were analysed using DOT-PLOT bi-parametric diagrams, using CELL QUEST Pro software (Becton Dickinson).
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