PBMCs were stained with CD4-FITC, CXCR5-PE, CXCR3-APC, CCR6-PE, CD27-FITC, CD38-PE, CD40L-PE, and IFNγ-PE antibodies (BDPharmingenTM, USA) and incubated at 4°C for 30 min in the dark. After the antigen–antibody incubation was completed, the specimen was cleaned 3 times with 2.5 mL of flowing washing solution (Hyclone, USA)/PBS, then resuspended in 500 μL of PBS, and put in storage at 4°C for performing upper flow cytometry analysis. The isotype control antibody was used to adjust the compensation of each channel and set the gate parameters. FlowJo software (Version 7.6.1, Tree Star Inc., USA) was employed to analyze the data obtained by flow cytometry. CD4+CXCR5+CXCR3+CCR6 indicates Th1-like cTfh cells, CD4+CXCR5+CXCR3-CCR6 indicates Th2-like cTfh cells, and CD4+CXCR5+CXCR3-CCR6+ indicates Th17-like cTfh cells.
Cxcr3 apc
Manufactured by BD
Sourced in United States
CXCR3-APC is a fluorescently-labeled monoclonal antibody that binds to the CXCR3 chemokine receptor. CXCR3 is a G protein-coupled receptor that is expressed on the surface of certain leukocytes and plays a role in their migration and activation. The APC (Allophycocyanin) fluorescent label allows for the detection and quantification of CXCR3-expressing cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Trustain fcx anti mouse cd16 32, by BioLegend (3 mentions)
Cd4 pe cf594, by BD (3 mentions)
Cd3 bv711, by BD (3 mentions)
Cd27 fitc, by BD (2 mentions)
Cd38 pe, by BD (2 mentions)
Il 10 apc, by BD (2 mentions)
Apc h2kd, by BioLegend (2 mentions)
Fitc nk1, by Thermo Fisher Scientific (2 mentions)
Pe cy7 tim3, by BioLegend (2 mentions)
Pd 1 pe, by BioLegend (2 mentions)
Cd8 bv510, by BD (2 mentions)
V450 cxcr4, by BD (2 mentions)
Ifnγ pe antibodies, by BD (1 mentions)
Cxcr5 pe, by BD (1 mentions)
Ccr6 pe, by BD (1 mentions)
Cd40l pe, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Ifn γ pe, by BD (1 mentions)
Cd27 pe cy7, by BD (1 mentions)
Foxp3 fitc, by BD (1 mentions)
11 protocols using cxcr3 apc
1
Multiparameter flow cytometry for cTfh subsets
2
Comprehensive Immune Cell Profiling
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The following monoclonal antibodies were purchased from eBioscience: CD278 (ICOS)-biotin, CD27-PeCy7, Foxp3-FITC, RORγt-PE, T-bet-PE; or from BD Biosciences: PD1-PECF594, CXCR3-APC, CD24-PECF594, CD25-BB515, CD44-PECy7, CD4-A700, CD8-A700, CD4-PB, CD62L-A700, GATA3-PE, RORγt-PECF594, STAT1 (pY701)-A488, IFNγ-PE, IL-10-APC, IL-17-PerCP-Cy5.5, streptavidin-PECy7. Live/dead fixable near-IR stain (Thermo Fisher) was used to exclude dead cells. For transcription factor staining, cells were stained for surface markers, followed by fixation and permeabilization before nuclear factor staining according to the manufacturer’s protocol (Foxp3 staining buffer set from eBioscience). For cytokine staining, cells were stimulated in media containing phorbol 12-myristate 13-acetate (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml, Sigma-Aldrich), and brefeldin-A (1/100, eBioscience) for 3 hr. After stimulation, cells were stained for surface markers, followed by fixation and permeabilization before intracellular staining according to the manufacturer’s protocol (cytokine staining buffer set from BD Biosciences). For phosphorylation staining, cells were stimulated with IFN-γ (50 ng/ml, PeproTech) for 30 min, fixed with formaldehyde, and permeabilized with methanol before staining. Flow cytometric analysis was performed on a Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star).
3
Purification and Characterization of Th17 Cells
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For Western blot and quantitative real-time PCR (qRT-PCR) analyses, untouched T cells were separated using the Pan T Cell isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of recovered cells, assessed by flow cytometer, was ≥97%.
For apoptosis and ERβ analyses of Th17 cells (i.e., CD4+CD45RA−CCR6+CXCR3−), CD4+ T cells were separated from PBMC by positive selection using CD4 MicroBeads (Miltenyi Biotec), with a purity ≥97%, as determined by flow cytometer. Then, CD4+CD45RA−CCR6+CXCR3− T cell subset was sorted by FACS (BD FACSAria; BD Biosciences) upon staining with the following mixture of mAb: CD4 PE/Cy7 (BD Biosciences), CD45RA FITC (BD Biosciences), CCR6 PE (Miltenyi Biotec), and CXCR3 APC (BD Biosciences). Sorted T cell subset was on average >95% pure as determined by postsorting flow cytometry analysis.
For apoptosis and ERβ analyses of Th17 cells (i.e., CD4+CD45RA−CCR6+CXCR3−), CD4+ T cells were separated from PBMC by positive selection using CD4 MicroBeads (Miltenyi Biotec), with a purity ≥97%, as determined by flow cytometer. Then, CD4+CD45RA−CCR6+CXCR3− T cell subset was sorted by FACS (BD FACSAria; BD Biosciences) upon staining with the following mixture of mAb: CD4 PE/Cy7 (BD Biosciences), CD45RA FITC (BD Biosciences), CCR6 PE (Miltenyi Biotec), and CXCR3 APC (BD Biosciences). Sorted T cell subset was on average >95% pure as determined by postsorting flow cytometry analysis.
4
Multiparametric Flow Cytometry of Leukocyte Subsets
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Absolute numbers and percentage of monocytes and lymphocyte subsets were determined with flow cytometry (FACS Calibur) on whole heparinized blood. Quantification beads (Trucount, BD) in combination with CD45 fluorescein isothiocyanate (FITC), CD14 phycoerythrin (PE), CD3 peridinin chlorophyll protein (PerCP) and CD19 allophycocyanin (APC) were used to measure absolute numbers of lymphocytes, monocytes, B cells and T cells according to the manufacturer’s instructions. Absolute numbers of cell subsets were calculated using the percentage of cells within a main cell type that was measured with quantification beads. For the subsets CD45 and CD3 or CD19 were always taken in multiple tubes combined with the following markers: CD8 PE, CD4 APC, CD16/56 PE, CXCR3 APC, CD80 PE and CD27 FITC (all products from BD Biosciences, San Jose, CA, USA). Flow cytometry data were analysed using FACSDIVA software version 6.1.3. Forward, sideward scatter and CD45bright were used to select lymphocytes. CD16+CD56+CD3− cells were defined as natural killer (NK) cells and CD16/CD56+ CD3+cells are expected to be predominantly CD56+CD16−. As CD56+CD3+ T cells, also described as NK T-like cells, have been described as activated effector cells [7 (link), 8 (link)] we define them here as activated T cells. Gating strategy is depicted in Additional file 1 : Figure S1.
5
Multiparameter Immune Cell Profiling
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CD3-percp-cy5.5 (Catalogue #45-0036-42), CD8-APC (Catalogue #17-0086-42), CD8-FITC (Catalogue #11-0086-42), CD14-APC (Catalogue #17-0149-42), CD56-FITC (Catalogue #4278380), CD16-PerCP-eFluor™710 (Catalogue #46-0168-42), CD11b-PerCP-eFluor™710 (Catalogue #46-0110-80), IFN-γ-PE (Catalogue #12-7319-42), TLR2-FITC (Catalogue #11-9922-41) and Mouse IgG1-PE (Catalogue #12-4714-81) were all bought from eBioscience. HLA-DR-PE (Catalogue #555812), IL-10-APC (Catalogue #554707), CXCR3-APC (Catalogue #550967) and IFN-γ-FITC (Catalogue #561053), Rat IgG2a-APC (Catalogue #554690) and mouse IgG-APC (Catalogue #5065947) were purchased from BD Biosciences. CD16-FITC (Catalogue #302006), HLA-DR-APC (Catalogue #307609), CD3-FITC (Catalogue #317306) and TLR4-APC (Catalogue #312815) were purchased from Biolegend. EP2-PE (Catalogue #10477) and Rabbit IgG-PE (Catalogue D5-1610) were purchased from Cayman Chemical.
6
Multiparametric Flow Cytometry Panel
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Cells were resuspended in PBS and stained on ice for 30 minutes in the dark with a fixable viability stain (BD Bioscience). Then, cells were resuspended into the stain buffer (BSA) (BD bioscience) and stained on ice for 30 minutes with various combinations of directly fluorochrome-conjugated antibodies. Antibodies used for flow cytometry were from BD Biosciences, Biolegend, or ThermoFisher, and include CD45 APC-Cy7 (clone 30-F11), CD8a BUV737 (clone 53-6.7), TCRb PE (clone H57-597), CD4 BUV395 (clone RM4-5), CD25 BV711 (clone PC61), CD11b AF700 (clone M1/70), Ly6G PercpCy5.5 (clone 1A8), CD11c PeCy7 (clone N418), Nkp46 FITC (clone 29A1.4), F4/80 PE-CF594 (clone T45-2342) Ly6C BV421 (clone AL-21), CXCR3 APC (clone CXCR3-173), Class I MHC FITC (clone 34-1-25) and PD-L1 BV421 (clone MIH5). For all samples, acquisition was performed on Fortessa flow cytometer (BD). Data were analyzed using FlowJo software (Tree Star).
7
Immunophenotyping of T cells from BAL and blood
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Dead cells were stained with BD Horizon™ Fixable Viability Stain 700. Moreover, the expression of markers on T cells from BAL fluid and blood was stained with antibodies, including anti‐human CD3‐APC/Cy7, CD25‐APC, CD4‐BB515, CXCR3‐APC, CCR4‐PE, CCR5‐APC, and CCR8‐PE (BD Biosciences). Cells were acquired with a flow cytometer (Beckman Coulter), and data were analyzed with FlowJo 10.
8
Multiparametric Flow Cytometry Immunophenotyping
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The cells were then stained with a panel of anti-human antibodies conjugated with different fluorophores against various T cell markers and consisted of CD45 APC-H7, CD3 PE-Cy7, CD4 BV605, CXCR3 APC, CCR6 BUV737, CD25 PerCP-Cy5.5, CD127 FITC, HLA-DR BV421 and CD38 PE (BD Biosciences, USA). Supplementary Table S1 represents the respective clones and catalog details of these antibodies. A sequential staining of cells was performed for chemokine receptors (CXCR3, CCR6) at room temperature giving an interval of at least 5 min before addition of the next antibody as proposed by Jalbert et al.31 (link). Next, the cells in the stained sample and the FMO control tubes were stained with a master mix of antibodies prepared in Brilliant Violet Staining buffer (BV Buffer, BD Biosciences, Germany) for twenty minutes on ice. Later on, 2 mL of the FACS buffer was added to wash the cells and subsequently the cells were resuspended in approximately 500 µL of the FACS buffer. Hoechst 33258 dye (Sigma Aldrich, Germany), a water soluble fluorescent dye, was added (0.1 µg/10 µL) approximately one minute before acquisition of each tube to discriminate between live and dead cells30 .
9
Intracellular Immune Cell Analysis
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Intracellular staining was performed using FOXP3 Fixation/Permeabilization Buffer (Biolegend -421403) according to the manufacturer instructions.
Antibodies used in this study were the following: TruStain fcX (anti-mouse CD16/32) (Biolegend -101320), CD3-BV711 (BD -563123), CD4-PECF594 (BD -562285), CXCR3-APC (BD -562266), CD44-V450 (BD -560451), Ki67-PECy7 (BD -561283), CD8a (KT15)-FITC (Proimmune 1705F/33790).
The data were acquired using BD LSR-FORTESSA flow cytometer and subsequently analyzed using FlowJo software v10.
Antibodies used in this study were the following: TruStain fcX (anti-mouse CD16/32) (Biolegend -101320), CD3-BV711 (BD -563123), CD4-PECF594 (BD -562285), CXCR3-APC (BD -562266), CD44-V450 (BD -560451), Ki67-PECy7 (BD -561283), CD8a (KT15)-FITC (Proimmune 1705F/33790).
The data were acquired using BD LSR-FORTESSA flow cytometer and subsequently analyzed using FlowJo software v10.
10
Multiparameter Flow Cytometry Analysis
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The antibodies were TruStain FcX anti-mouse CD16/32 (BioLegend), APC-H2Kd (BioLegend), BV711-CD3 (BD Horizon), PE-CF594-CD4 (BD Horizon), FITC-NK1.1 (Invitrogen), PE-PD1 (BioLegend), APC-CXCR3 (BD Pharmigen), PE-CY7-TIM3 (BioLegend), BV510-CD8 (BD Horizon), and V450-CXCR4 (BD Horizon).
The data were acquired using BD LSRFortessa flow cytometer and analyzed using FlowJo software v9 (Ashland, OR).
The data were acquired using BD LSRFortessa flow cytometer and analyzed using FlowJo software v9 (Ashland, OR).
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