The immunophenotyping of hBM-MSC unlabeled and labeled with 50 µg Fe/mL of MFNP during 24 h (the maximum incubation time analyzed in this study) was performed using the following antibodies: CD19-FITC (clone:4G7) and CD34-PE (clone: 8G12) from BD Biosciences, San Jose, CA, USA; CD14-Alexa 700 (clone: M5E2), CD29-APC (clone: MAR04), CD35-FITC (clone: E11), CD44-PerCP-Cy5.5 (clone: G44-26), CD73-PE (clone: AD2), CD90-PE-Cy7 (clone: 5E10), HLA-DR-APC-H7 (clone: G46-6), CD106-FITC (clone: 51-10C9) all from BD Pharmingen, San Diego, CA, USA; CD31-V450 (clone: WM59), CD45-V500 (clone: H130), CD105-PE-CF546 (clone: 266) all from BD Horizon, San Jose, CA, USA, unstained samples and fluorescence minus one (FMO) was used as a fluorescence background control. Briefly, 1 × 105 cells were incubated in the dark/room temperature for 30 min in the presence of antibodies at concentrations recommended by the manufacturers. Cells were then washed with a buffered solution, and at least 10,000 events were acquired using FACS LSRII FORTESSA equipment (BD Biosciences). Data analyses were performed using FlowJoTM software (BD Biosciences), and results were presented as graphics where the FMO is overlaid with the stained sample for each antibody/marker used.
Cd44 percp cy5
Manufactured by BD
Sourced in United States
CD44-PerCP-Cy5.5 is a fluorescently-labeled antibody that binds to the CD44 cell surface receptor. It is used for the detection and analysis of CD44-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd73 pe, by BD (4 mentions)
Cd4 pb, by BD (4 mentions)
Cd62l apc, by BD (4 mentions)
Cd29 apc, by BD (3 mentions)
Cd62l fitc, by BD (3 mentions)
Cd8 apc cy7, by BD (3 mentions)
Cd127 pe, by BD (3 mentions)
Tissue culture grade plastic ware, by BD (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Ifn γ pe cy7, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cd45 v500, by BD (3 mentions)
F4 80 apc, by BD (2 mentions)
Cd40 pecy5, by BD (2 mentions)
Cd86 pe, by BD (2 mentions)
Abs for elisa, by BD (2 mentions)
Klrg1 pe, by BD (2 mentions)
Tnfα percpcy5, by BD (2 mentions)
Il 17 percpcy5, by BD (2 mentions)
Cd105 alexafluor488, by Bio-Rad (2 mentions)
12 protocols using cd44 percp cy5
1
Immunophenotyping of hBM-MSC with MFNP
2
Phenotypic Analysis of T Cells
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For phenotypic analysis of T cells, the PPD and peptides stimulated lungs and spleen/LNs cells were analysed by flow cytometry. Lymphocytes culture were harvested and stained with fluorochrome tagged anti-CD4-PE, CD8-APCCy7, CD62L-FITC, CD44-PerCPCy5.5, CD11c-PECy7, F4/80-APC, CD86-PE, CD80-FITC, CD40-PECy5, and MHC-II-PerCPCy5.5abs (BD Biosciences, San Jose, CA). Briefly, lymphocytes were harvested in tubes and washed with FACS buffer (PBS + 2%FCS). Cells were Fc blocked using anti-mouse CD16/CD32 Ab. Later, stained with fluorochrome-labelled Abs. After staining, cells were fixed by using 1% paraformaldehyde in FACS buffer. Cells were acquired in BD-FACS Aria III and BD-FACS Accuri (BD, Franklin Lakes, NJ). The analysis was performed using BD-FACS DIVA, BD-C6, and Flowjo software (BD, Franklin Lakes, NJ).
3
Mouse T Cell Phenotyping Protocol
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All chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO, United States) unless otherwise mentioned. For culturing of cells, tissue culture grade plastic ware was purchased from BD Biosciences (Bedford, MA, United States). RPMI-1640 media and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, United States) for cell culture. L-pyruvate, L-glutamine, streptomycin and penicillin were from Serva (Heidelberg, Germany). Anti–mouse fluorochrome labeled antibodies (Abs): CD4-PB, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, and Abs for ELISA were procured from BD Pharmingen (San Diego, CA, United States) or otherwise mentioned.
4
Splenocyte Isolation and Analysis
5
Detailed Protocols for Immune Cell Analysis
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All standard chemicals and reagents used in the study were purchased from Sigma (St. Louis, Mo., USA), and fluorochrome labeled Abs to CD4-FITC, CD8-APCCy7, CD44-PerCPCy5.5, CD62L-APC and KLRG1-PE were obtained from BD Biosciences (San Diego, CA), unless indicated. RPMI-1640 and FCS were purchased from GIBCO (Grand Island, NY). For culturing of cells, tissue culture grade plastic-ware was purchased from BD Biosciences (Bedford, MA). Purified protein derivative (PPD) of Mtb was prepared by standard protocols, as described elsewhere [47 (link), 48 (link)].
6
Characterization of Human iPSCs and iPSC-MSCs
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Cell surface markers and pluripotency markers were analyzed for human iPSCs and iPSC-MSCs. We incubated 1.5 × 105 cells with each of the following conjugated monoclonal antibodies: CD44-PerCP-Cy5.5, CD144-PerCP-Cy5.5, CD90-PE, CD73-PE, CD166-PE, CD105-PE, CD34-APC, CD45-APC, OCT3/4-PE, SOX-2-Percp-Cy5.5, TR-181-PE, and TRA-161-Percp-Cy5.5 (BD Bioscience, San Diego, CA, USA), and analyzed with fluorescence-activated cell sorting (FACS). For the analysis of the nuclear pluripotency markers OCT3/4 and SOX-2, iPSCs were first ruptured by the Transcription Factor Fixation/Permeabilization Concentrate Kit (eBioscience, USA), and then fluorescent staining was performed. Nonspecific fluorescence was determined by incubation of similar cell aliquots with isotype-matched antibodies (BD Bioscience). Data were analyzed using the Beckman Coulter Gallios flow cytometer (Fullerton, CA, USA). Analysis was performed using FCS3.0 and Kaluza software (Beckman Coulter).
7
Macrophage Polarization Modulation
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All standard chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, United States) unless otherwise mentioned. ELISA antibodies, recombinant cytokines, and fluorochrome tagged antibodies for flow cytometry: F4/80-APC, CD11b-PerCP-Cy5.5, CD40-PE-Cy5, CD86-PE, MHC-II-PerCPefluor710, Annexin-FITC, CD4-PB, IL-17-PE, IFN-γ-PE-Cy7, CD62L-FITC, CD44-PerCP-Cy5.5, and CCR7-PECy7 are procured from BD Biosciences (San Diego, CA, United States). Antibodies for western blot analysis against: β-actin, goat anti-rabbit IgG-HRP, and donkey anti-mouse IgG-HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States); iNOS, pSTAT-1 (pY701), STAT-1, pSTAT-3 (pY705), STAT-3, pSTAT-6 (pY641) and STAT-6 antibodies were procured from Cell Signaling Technology (Danvers, MA, United States). Inhibitors used in the experiments such as STAT-1 inhibitor (fludarabine), Syk inhibitor (piceatannol) and iNOS inhibitor (NM, NG-Monomethyl-L -arginine) are from Calbiochem (Billerica, MA, United States). Curdlan was purchased from InvivoGen (San Diego, CA, United States). All plastic-ware of tissue culture grade was procured from BD Biosciences (Bedford, MA, United States).
8
Comprehensive Immunophenotyping Protocol
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Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse or anti-human fluorochrome labeled antibodies (Abs): CD4-PB, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, FoxP3-FITC, Tim3-PE, PD1-PECy7, IFN-γ-PECy7, TNFα-PerCPCy5.5, IL-17-PerCPCy5.5, CD25APC-Cy7, CD45RA-PE, CD45RO-APC, and Abs for ELISA were procured from BD Pharmingen (San Diego, CA) or otherwise mentioned. RPMI-1640 and FBS were purchased from GIBCO (Grand Island, NY) for cell culture. For culturing of cells, tissue culture grade plastic-wares were purchased from BD Biosciences (Bedford, MA). Ab against iNOS used in Western blot was procured from (Abcam, Cambridge, United Kingdom).
9
Quantifying Cellular Marker Expression and Uptake
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The expression of cell surface markers was quantified by flow cytometric analysis. Stromal cells were labelled with antibodies CD29-APC, CD44-PerCP-Cy5.5, CD45-V500, CD73-PE, CD117-PE-Cy7, PerCP-Cy5.5 CD90 (BD Biosciences, San Jose, USA), and CD105-AlexaFluor488 (AbD Serotec, Oxford, UK). Respective isotype antibodies served as negative controls. A measurement of 3 × 104 events was carried out using BD FACS LSRII flow cytometer (BD Biosciences).
To evaluate miRNA and mRNA uptake efficiency, cells were treated with different amounts of Cy3-labeled Pre-miRNA Negative Control #1 (AM17120, Thermo Fisher) or GFP-mRNA (Trilink, San Diego, USA) and analyzed by flow cytometry 24 h post transfection. To detect cytotoxicity, cells were labelled with Near-IR LIVE/DEAD fixable dead cell stain kit (Molecular Probes, Eugene, USA). Analysis of flow cytometry data, including gating, was conducted with the FACSDiva software, Version 8. (Becton Dickinson).
To evaluate miRNA and mRNA uptake efficiency, cells were treated with different amounts of Cy3-labeled Pre-miRNA Negative Control #1 (AM17120, Thermo Fisher) or GFP-mRNA (Trilink, San Diego, USA) and analyzed by flow cytometry 24 h post transfection. To detect cytotoxicity, cells were labelled with Near-IR LIVE/DEAD fixable dead cell stain kit (Molecular Probes, Eugene, USA). Analysis of flow cytometry data, including gating, was conducted with the FACSDiva software, Version 8. (Becton Dickinson).
10
Characterization of hMSC Surface Markers
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Cell surface markers of freshly isolated and cultured CD105+ hMSCs were fluorescently labeled with anti-human antibodies CD29-APC, CD44-PerCP-Cy5.5, CD45-V500, CD73-PE, CD117-PE-Cy7 (BD Biosciences, Heidelberg, Germany), and CD105-AlexaFluor488 (AbD Serotec, Kidlington, UK). Respective mouse isotype antibodies served as negative controls. 3 × 104 events were acquired using BD FACS LSRII flow cytometer (BD Biosciences) and analyzed with BD FACSDiva Software 6 (BD Biosciences).
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