Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.
Cd45ro fitc
Manufactured by BD
Sourced in United States
CD45RO-FITC is a fluorescently labeled antibody that binds to the CD45RO isoform of the CD45 protein. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of various hematopoietic cells. The CD45RO isoform is a marker for memory T cells.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Ccr7 pe cy7, by BD (3 mentions)
Ccr7 pe, by BD (2 mentions)
Cd8 percp, by BD (2 mentions)
Cd4 efluor450, by Thermo Fisher Scientific (2 mentions)
Cd45ro pe cy7, by BD (2 mentions)
Facscanto clinical software, by BD (2 mentions)
Multitest trucount method, by BD (2 mentions)
Lymphoprep, by Axis-Shield (2 mentions)
Cd3 apc, by BD (2 mentions)
Cd56 pe cy7, by BD (2 mentions)
Cd19 pe, by BD (2 mentions)
Monensin, by BD (2 mentions)
Count and viability kit, by Merck Group (2 mentions)
Muse cytometer, by Merck Group (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Cd16 kromeorange, by BD (2 mentions)
Cd107a hv500, by BD (2 mentions)
Cd45ra apcalexafluor750, by BD (2 mentions)
Cd31 pe, by BD (2 mentions)
9 protocols using cd45ro fitc
1
Phenotyping of Immune Cells by Flow Cytometry
2
Comprehensive Immune Cell Profiling by Flow Cytometry
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Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation with Lymphoprep (Axis-Shield). PBMC or whole blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-efluor605, CD4-efluor450, CD27-APC-efluor780, HLA-DR-efluor780, CD45RA-efluor605, FOXP3-PE (eBioscience), CD4-APC-H7, CD8-Percp, CD8-PE-Cy7, CD31-AF647, CD45RO-FITC, CD45RO-PE-Cy7, CCR7-PE-Cy7, Ki-67-Percp-cy5.5, CTLA-4-BV421 (BD Biosciences), PD-1-PE, CD28-AF700 (Biolegend), and CD161-PE (Miltenyi Biotec). Intracellular staining for FOXP3, Helios, Ki-67, and CTLA-4 was performed after cells were permeabilized with a FOXP3 staining buffer set according to instructions of the manufacturer (eBioscience). Whole blood samples were treated with BD lysing solution according to the instructions of the manufacturer (BD Biosciences). Stained samples were analyzed on a LSR-II flow cytometer (BD Biosciences). Analysis was performed with Kaluza Flow Analysis Software (Beckman Coulter). Absolute numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells were determined according to the MultiTest TruCount method (BD Biosciences), as described by the manufacturer. TruCount samples were measured on a FACSCanto-II (BD Biosciences) and analyzed with FACSCanto Clinical Software (BD Biosciences).
3
Multicolor Flow Cytometry for T Cell Subsets
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PFMC or PBMC were stained with LIVE/DEAD yellow fixable dead cell stain kit (Life Technology, Eugene, OR) and with the following two monoclonal antibody staining panels: In Panel 1, cells were surface-stained by anti-CD3-PerCP, CD4-PE-Cy7, CD45RO-FITC, CD69-APC, CD38-AF700 (BD Bioscience, San Jose, CA), CD8-PE-TR (Invitrogen, Frederick, MD), CCR7-APC-Cy7 and HLADR-Pacific Blue (BioLegend, San Diego, CA). Cells were then fixed, permeabilized, and washed with Transcription Factor Buffer Set (BD Pharmingen) according to the manufacturer and stained with anti-HIV-1 p24-PE (KC57) (Beckman Coulter, Indianapolis IN). In Panel 2, monoclonal antibodies included: anti CD25-APC (BioLegend), CCR5-V450 and intracellular staining for Ki67-BV711 (both from BD Bioscience) in addition to anti-CD3, CD4, CD8, CCR7, CD45RO, and HIV-1 p24 as in panel 1. Gates were set using Fluorescent-Minus-One controls for each sample. T cells were identified as naïve (CD45RO-CCR7+), central memory (Tcm) (CD45RO+CCR7+), effector memory (Tem) (CD45RO+CCR7-), and terminally differentiated effector memory (TemRA) (CD45RO-CCR7-). Stained samples were analyzed by a LSRII cytometer (BD). Data were analyzed using FlowJo (Tree Star, Ashland, OR).
4
NK Cell Degranulation Assay Protocol
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This was done as previously described (15 (link)). Briefly, 50 × 103 target cells per well were placed in RPMI, 10% FBS, IL-2 100 U/mL with monensin (BD Biosciences) in a 96-well V-bottom plate. NK and target cells were incubated overnight at 37°C in 5% CO2, and living cells were counted using a Muse cytometer (Millipore) with a count and viability kit (Millipore). As a control, NK cells were incubated without target cells. CD107a+ NK cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using 7AAD, CD45RO-FITC, CD19-PE, CD56-PECy7, CD3-APC, CD45RA-APCAlexaFluor750, CD16-KromeOrange, and CD107a-HV500 (BD Biosciences). The results were analyzed using Kaluza software.
5
Comprehensive T-cell Immunophenotyping
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Isolated mononuclear cells or whole blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies: CD4-PcP, CD8-APC-H7, CD31-PE, CD45RA-FITC, CD25-PE, CD45RO-PE-Cy7, CD45RO-FITC, CCR7-PcP-Cy5.5, TCRγδ-BV421, pSTAT5-PE-Cy7, Ki-67-PcP-Cy5.5, Tbet-PE, CXCR3-PE-Cy5, CD69-APC-Cy7, CCR4-PE-Cy7, CD5-APC, CRTH2-PE, GATA3-APC, CD5-APC (all BD), CD4-ECD, CD4-PC7, CD69-PC5, CD69-ECD, Beta Mark TCR V β kit (all Beckman Coulter, Woerden, The Netherlands), CD122-PE, CD132-PE, CCR6-PcP-Cy5.5, IL-2-AF700, IL-4-PE, IFN-γ-PcP-Cy5.5, FOXP3-AF647, Helios-AF488 (all Biolegend, Uithoorn, The Netherlands), CD4-ef450, CD25-APC, CD25-PE, CD45RA-PE, CD45RA-ef605, HLA-DR-APC-ef780, IL-17-AF488, CD27-AF700, CD28-PcP-cy5.5 (all eBioscience, Vienna, Austria). In case of whole blood staining, samples were lysed with BD FACS lysing solution. Samples were measured on a LSR-II (BD) or FC500 (Beckman Coulter) and analyzed with Kaluza Analysis Software (Beckman Coulter). Absolute numbers of CD4+ and CD8+ T cells were determined according to the BD MultiTest TruCount method, as described by the manufacturer. TruCount measurements were taken on a FACS Canto-II (BD) and analyzed with FACSCanto Clinical Software (BD).
6
PBMC Isolation and T-Cell Subpopulation Sorting
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Peripheral blood was collected in heparin-containing vacutainer tubes (Becton Dickinson, Franklin Lakes, USA) and peripheral blood mononuclear cells (PBMC) were freshly isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway) according to the manufacturer’s protocol. CD4 T cells were sorted by fluorescence-activated cell sorting (FACS) as CD3+CD4+ and CD8 T cells as CD3+CD4-. Within the CD4 and CD8 T-cell subsets, naïve (CD45RO-) and memory (CD45RO+), truly TNAIVE (CD45RO-CCR7+) and terminally differentiated (TEMRA) cells (CD45RO-CCR7-); as well as CD3+ and CD31- populations were isolated using combinations of the following anti-human monoclonal antibodies: CD3-e450, CD4-A647 (eBioscience, Vienna, Austria), CD45RO-FITC, CCR7-PE, CCR7-PECY7 and CD31-PE (BD Bioscience, Breda, The Netherlands). See Figure A in in S1 File for sorting schemes.
7
NK Cell Cytotoxicity Assay
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Briefly, 50×103 target cells per well were placed in RPMI, 10% FBS, IL-2 100 U/mL with monensin (BD Biosciences) in a 96-well V-bottom plate. NK and target cells were incubated overnight at 37°C in 5% CO2 and living cells were counted using a Muse cytometer (Millipore) with the count and viability kit (Millipore). As a control, NK cells were incubated without targets. CD107a+ NK cells were analyzed on a Gallios flow cytometer (Beckman Coulter) using 7AAD, CD45RO-FITC, CD19-PE, CD56-PECy7, CD3-APC, CD45RA-APCAlexaFluor750, CD16-KromeOrange and CD107a-HV500 (BD Biosciences). Results were analyzed using Kaluza software.
8
Flow Cytometry of Memory T Cell Subsets
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Samples of fresh ACD-anticoagulated whole blood were stained with monoclonal antibodies, lysed and fixed, as above. Monoclonal antibodies used to study subsets of CD4 memory T cells were: CD3-PerCP-Cy5.5, CXCR3-Alexa Fluor 488 and -PE-CF594, integrin ß7-APC, CD4-Alexa Fluor 700, CD45RO-FITC, CD45RA-PE-CF594; CD49d-PE, CD25-PE-Cy5, CD8-BV786 (BD Biosciences), CD45RO-ECD (Beckman Coulter); CD49d-BV510, integrin ß7-biotin, CCR6-BV421, CD127-PE-Cy7 (BioLegend); CD161-PE, -APC or PE-Vio-770 (Miltenyi Biotec, Bergisch Gladbach, Germany); and CD62 L-APC-Cy7 (eBiosciences). A total of 400,000 events were collected, and a manual forward and side scatter gate was drawn to include lymphocytes, then gated sequentially on CD31, on CD41, and then on CD45RO1 cells using FlowJo, then exported as an FCS file using FlowJo.
9
Immunophenotyping of T and B Cells
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Blood samples were washed and incubated with antihuman CD3-AlexaFluor700, CD4-eFluor450 (eBioscience, San Diego, USA), CD45RO-FITC, CCR7-PE-Cy7 (BD Biosciences, Franklin Lakes, USA), CXCR3-APC-Cy7, CCR4-PerCP-Cy5.5 and CCR6-BV605 (BioLegend, San Diego, USA) to determine CD45RO + CCR7 -CCR6 + CCR4 + CXCR3 À Th EM 17 cells and CD45RO + CCR7 À CCR6 À CCR4 À CXCR3 + Th EM 1 cells [2, 9] , or anti-human CD19-eFluor450, CD38-PE-Cy7 (eBioscience) and CD24-FITC (BD Biosciences) to determine CD24 hi CD38 hi Bregs. Samples were fixed, washed and acquired on a LSR-II (BD Biosciences). For gating strategies see Supplementary Fig. S1, available at Rheumatology online.
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