Flow cytometry was conducted using an LSRFortessa (BD Biosciences). A panel consisting of antibodies conjugated to six different fluorophores was used to classify subsets of memory T cells and for drop-seq. Antibodies used were: CD8α-Pacific blue (BioLegend), CD3-BV650 (BD Biosciences), CD45RA-APC-Cy7 (BioLegend), CCR7–488 (Bio-Legend), IL-7Rα-PE (BioLegend) and CD27-PE-Cy7 (BioLegend). For characterization of CSF cells, this same panel was used, but CD19-PE-Cy5 (BioLegend) and CD14-Qdot-705 (Thermo Fisher Scientific) were included. For sorting CSF T cells for TCR plate-seq, the following antibodies were used: CD8α-PE (BioLegend), CD161-PE-Cy7 (BioLegend), CXCR3-APC (BioLegend), CD4-APC-Alexa700 (Thermo Fisher Scientific), CD39-APC-Cy7 (BioLegend), CD38-FITC (BioLegend), PD-1-BV421 (BD Biosciences), CD45RA-BV605 (BD Biosciences), CD3-BV650 (BD Biosciences), CD27-BV786 (BD Biosciences) and CD127-BUV395 (BD Biosciences). For each experiment, a compensation matrix was developed using singly stained and unstained controls or fluorescent beads, and all analysis was conducted in Cytobank.
Cxcr3 apc
Manufactured by BioLegend
Sourced in United States
CXCR3-APC is a fluorochrome-conjugated antibody that binds to the CXCR3 receptor. CXCR3 is a chemokine receptor that is expressed on the surface of various cell types, including T cells, natural killer cells, and dendritic cells. The APC (Allophycocyanin) fluorochrome allows for the detection and analysis of CXCR3-expressing cells using flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Ccr4 pe cy7, by BioLegend (2 mentions)
Cd8α pacific blue, by BioLegend (1 mentions)
Pd 1 bv421, by BD (1 mentions)
Cd38 fitc, by BioLegend (1 mentions)
Cd27 pe cy7, by BioLegend (1 mentions)
Cd19 pe cy5, by BioLegend (1 mentions)
Cd45ra apc cy7, by BioLegend (1 mentions)
Cd8α pe, by BioLegend (1 mentions)
Cd27 bv786, by BD (1 mentions)
Cd127 buv395, by BD (1 mentions)
Cd45ra bv605, by BD (1 mentions)
Cd3 bv650, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cd45ra fitc, by BioLegend (1 mentions)
Ccr6 bv421, by BioLegend (1 mentions)
Cd4 percp, by BioLegend (1 mentions)
Anti cd3 anti cd28 beads, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cd3 percp cy5, by BioLegend (1 mentions)
6 protocols using cxcr3 apc
1
Multiparameter Flow Cytometry of CSF Immune Cells
2
Analyzing IL-17 Positive Th17 Cells
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IL-17 positive Th17 cells were analyzed with accepted methods. Briefly, fresh PBMCs were stimulated for 16 hours with anti-CD3/anti-CD28 beads (Life Technologies) in the presence of Brefeldin A (Sigma-Aldrich). Thereafter, the cells were fixed, permeabilized and stained with anti-CD4, -CD69-APC, and -IL-17A (BD Biosciences), analyzed with BD LSRFortessa flow cytometer (BD Biosciences) and using FlowJo software (BD Biosciences). Th17 CD4+ memory cells were detected from whole blood by a four-color flow cytometry panel with monoclonal antibodies (mAbs) [CD45RA-FITC, CD4-PerCP, CXCR3-APC and CCR6-BV421 (BioLegend)] against surface antigens.
3
Multi-parameter Flow Cytometry Analysis
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Surface staining was performed on cells washed with FACS buffer (PBS, 2% FCS, 0.05% sodium azide, 0.5 mM EDTA). Dead cells were detected with Zombie NIR™ Fixable Viability Kit (Biolegend) according to the manufacturer’s protocol. Cells were then stained for 20 minutes at room temperature with antibody mixes including the following: CD3-PeCy7, CD3-PerCPCy5.5, CD4-FITC, CD4-PECy7, CD8-PerCPCy5.5, CD14-PerCPCy5.5, CD16-FITC, CD45RO-PE, CD45RA-APC, CCR6-PE, CXCR3-APC, and HLA-DR-PECy7 (all from Biolegend). For intracellular staining, cells were washed after surface staining, fixed with 2% PFA for 15 minutes at RT, and washed with permeabilization buffer (FoxP3 eBioscience buffer). Cells were then intracellular stained for 30 minutes at RT with antibody mixes that were made in permeabilization buffer, including interferon (IFN)γ-PB and IL-4-AF647 (all from Biolegend). Cells were washed with FACS buffer and acquired using a BD Canto II. All experiments were analysed using FlowJo v10.4 (Becton, Dickinson and Company, Ashland, OR, USA).
4
Cytokine Analysis of Splenic Cells
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For cytokine analysis, 2 x 106 splenic cells were plated into a 96-well plate. Cells were suspended in RMPI 1640 culture medium and incubated for four hours utilizing phorbol 12-myristate 13-acetate (30ng/mL) and ionomycin (400ng/mL) with 10μg/mL of Brefeldin A at 37°C. After stimulation, cells were then stained for the following intracellular and extracellular markers CD4—Pacific Blue (BD Biosciences, clone RM4-5), CD8—Pacific Orange (Invitrogen, clone MCD0830), CD25—FITC (BioLegend, clone PC61), CD3—Alexa 700 (BD Biosciences, 500A2), FOXP3—APC (eBioscience, FJK-16S), IL-2—FITC (BD Pharmingen, clone JES6-5H4), IL-4—PE (BioLegend, clone 11B11), CCR4—PE Cy7 (BioLegend, clone 2G12), CXCR3—APC (BioLegend, clone CXCR3-173), and IL-17A—PE (eBioscience, clone eBio17B7). Samples were run on an LSR II flow cytometer (BD Biosciences) and subsequent data was analyzed with FlowJo 10.0.8rl software (Tree Star, San Carlos, CA).
5
Immunophenotyping of Chronic Lymphocytic Leukemia
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PBMCs were separated by density centrifugation of fresh CLL blood samples. PBMCs were incubated with directly conjugated mAbs for 20 minutes at room temperature. Flow cytometric analysis was performed after erythrolysis with IOTest® 3 Lysing Solution (Beckman Coulter, IM3514) and washing steps. Monoclonal antibodies for analysis were provided by Biolegend: anti-human PD-1-Brilliant Violet 421 (329920),CXCR3-APC (353708), CXCR5-PerCP-Cy5.5 (356910), CCR4-PE-Cy7 (359410), CD62L-PerCP/Cy5.5 (304824) and CD226-PerCP/Cy5.5 (338314). Monoclonal antibodies provided by ThermoFisher included TIGIT-PE (12–9500-42), CD4-PE-Cyanine7 (25–0048-42), CD4-eFluor450 (48-0048-42), CD4-APC (MHCD0405), 2B4-APC (17-5837-42), CD3-Alexa Fluor 700 (56-0032-82), CD3-FITC (11-0039-42), CD45RA-APC-eFluor780 (47-0458-42), CD25-PE-Cyanine7 (25-0259-42), CD127-APC-eFluor780 (47-1271-82), CD155-PE (12-1550-41), CD112-APC (17-1128-42), CD19-FITC (11-0199-42), rhAnnexin V-FITC (BMS147FI), 7-AAD Viability Staining Solution (00-6993-50), TIM-3-PerCP-eFluor 710 (46-3109-42) and CD8-Pacific Orange (MCD0830). For viability assays, rhAnnexin V/FITC, 7-AAD Viability Staining Solution was used. Data acquisition was performed on Gallios™ Flow Cytometer research system (Beckman Coulter) and data analysis was performed using Kaluza 1.2 Flow Cytometry Analysis Software (Beckman Coulter).
6
Multicolor Flow Cytometry Analysis
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For the flow cytometry, the cells were washed with PBA (PBS supplemented with 0.5% bovine serum albumin (BSA), 1% FBS, and 0.1% sodium azide), and incubated with conjugated antibodies against surface markers (CD3-CF (Immunostep, Salamanca, Spain); CD56-PC5 (Phycoerythrincyanin 5), CD16-PE-Cy7 (phycoerythrin-Cy7), CD14-APC (Allophycocyanin), CD15-PB (Pacific Blue), and CXCR3-APC (BioLegend, San Diego, California, USA) at 4 °C for 30 minutes in the dark. For intracellular staining, after surface labelling, the cells were fixed with 1% p-formaldehyde for 10 min at room temperature (RT), permeabilized with 0.2% saponin for 10 min at RT, and stained with anti-CXCL10-PE (BD, San Jose, California, USA) or mouse IgG2a-PE (Beckman Coulter, Brea, California, USA) for 30 min. The cells were washed in PBA and analyzed using a Gallios Flow Cytometer (Beckman Coulter, Brea, California, USA). The analysis of the experiments was performed using either Kaluza (Beckman Coulter, Brea, California, USA) or FlowJo software (Ashland, Oregon, USA, https://www.flowjo.com ).
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