Anti cxcr3 apc

Manufactured by BD

Sourced in United States

Anti-CXCR3 APC is a fluorescently-labeled antibody that binds to the CXCR3 chemokine receptor. CXCR3 is a G protein-coupled receptor expressed on the surface of certain immune cells, such as T cells and NK cells.

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Lab products found in correlation

Anti cd4 pe cy7, by BD (4 mentions) Anti pd 1 pe, by BD (2 mentions) Anti cd8 apc cy7, by BD (2 mentions) Fluo 3, by Thermo Fisher Scientific (2 mentions) Anti ccr4 pe, by BD (2 mentions) Anti cd8 percp, by BD (1 mentions) Anti ki67 fitc, by BD (1 mentions) Anti cd62l pe, by BD (1 mentions) Anti cd3 pe cy7, by BioLegend (1 mentions) Foxp3 staining buffer, by Thermo Fisher Scientific (1 mentions) Brefeldin a, by Merck Group (1 mentions) Anti t bet pe ebio4b10, by Thermo Fisher Scientific (1 mentions) Anti cd127 pe cy7, by BD (1 mentions) Anti cd122 fitc, by BD (1 mentions) Anti cd90.2 pe cy7, by BD (1 mentions) Cyan flow cytometer, by Agilent Technologies (1 mentions) Facs lysing buffer, by BD (1 mentions) Anti nk1.1 fitc, by BD (1 mentions) Anti bcl2 pe, by BD (1 mentions) Canto 2, by BD (1 mentions)

Spelling variants of this product

CXCR3-APC (11 citations) Anti-CXCR3 (5 citations)

9 protocols using anti cxcr3 apc

Multiparameter Flow Cytometry of T-cell Subsets

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Purified CD8⁺ T cells, splenocytes and thymocytes were first stained for surface antigens and then treated with Foxp3 staining buffer set according to the manufacturer's directions (eBioscience). Anti-Eomes AlexaFluor 647 or eFluor 660 (Dan11mag, 1/75), anti-T-bet PE (eBio4B10, 1/100) and anti-CD49d FITC or PE (R1-2, 1/50) antibodies were purchased from eBioscience. Anti-CD8 PercP (53–6.7, 1/50), anti-CXCR3 APC (CXCR3-173, 1/50), anti-CD4 Pe-Cy7 (RM4-5, 1/100), anti-CD62L PE (1/100) or V450 (1/50) (MEL-14), anti-Bcl2 PE (3F11, 1/25), anti-Ki67 FITC (B56, 1/25), anti-CD44 FITC or V450 (IM7, 1/50), anti-CD127 Pe-Cy7 (SB/199, 1/50), anti-CD122 FITC (TM-BETA1, 1/50), anti-NK1.1 FITC (PK136, 1/50), anti-CD90.2 Pe-Cy7 (53-2.1, 1/100) and anti-IFNγ APC or PB or PE (XMG1.21/50) were purchased from BD biosciences. Anti-CD3 Pe-Cy7 (2C11, 1/100) was purchased from Biolegend.
In some experiments, brefeldin A (5 μg ml⁻¹, Sigma) was added in samples for 3 h at 37 °C before intracytoplasmic staining. Blood samples were directly stained for surface antigens and then treated with FACS lysing buffer (BD biosciences) as described in the product data sheet. All samples were fixed with 1% paraformaldehyde in PBS prior to their processing using a Cyan flow cytometer (Dako Cytomation).

Martinet V., Tonon S., Torres D., Azouz A., Nguyen M., Kohler A., Flamand V., Mao C.A., Klein W.H., Leo O, & Goriely S. (2015). Type I interferons regulate eomesodermin expression and the development of unconventional memory CD8+ T cells. Nature Communications, 6, 7089.

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NK Cell Surface Marker Analysis

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Staining and flow cytometric analysis was performed as described before [10 (link)]. The Following monoclonal antibodies were used in this study: Via probe PerCP, anti-CD19 PerCP, anti-CD14 PerCP and anti-CD3 PerCP (BD Biosciences, CA, USA) to exclude dead cells, B cells, monocytes and T cells, respectively, and anti-CD56 PC7 (Beckman Coulter, CA, USA) and anti-CD16 APC-H7 (BD Biosciences, CA, USA) to identify NK cells. Additional antibodies that were used include: anti-CXCR3 APC (BD Biosciences, CA, USA), anti-CD69 PE (Invitrogen, CA, USA), anti-CD127 PacBlue (BD Biosciences, CA, USA), anti-CD95 APC (Biolegend, CA, USA), anti-NKp30 APC (Beckman Coulter, CA, USA), anti-NKp46 PE (Beckman Coulter, CA, USA), anti-NKG2D APC (BD Biosciences, CA, USA). At least 1 million events were acquired for each sample, using the BD Canto II (BD Biosciences, CA, USA). Data were analysed with FlowJo 8.8.4 (TreeStar, Or, USA). Lymphocytes were defined by forward and side scatter. CD3⁺, CD14⁺, CD19⁺, dead cells and cell aggregates were removed from analysis based on PerCP and Viaprobe cell viability staining and pulse width analysis. Fluorescence minus one (FMO) staining was used to determine threshold values for the expression of each surface marker.

Bhardwaj S., Ahmad F., Wedemeyer H., Cornberg M., Schulze zur Wiesch J., van Lunzen J., Sarin S.K., Schmidt R.E, & Meyer-Olson D. (2016). Increased CD56bright NK cells in HIV-HCV co-infection and HCV mono-infection are associated with distinctive alterations of their phenotype. Virology Journal, 13, 67.

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HLA Class II Tetramer Staining Protocol

Cited 1 time

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Direct HLA class II tetramer staining was performed essentially as previously described (11 , 12 (link)), except that cells were labeled with PE and PE-CF594 labeled tetramers. Briefly, 30×10⁶ PBMCs were re-suspended in 200 µL of T cell media, incubated with 50 nM dasatinib for 10 minutes at 37°C, and stained with PE-DRB4/PPI 9–28 tetramer and PE-CF594-DR0401/PPI 76–90 (with an Lys Ser substitution at position 88, as described (13 (link))) at room temperature for 100 minutes. Cells were washed, incubated with anti-PE magnetic beads (Miltenyi) for 20 minutes at 4°C and enriched with a magnetic column, retaining 1% of the cells as a non-enriched sample. The enriched and pre-column samples were labeled with anti-CD4 PerCP-Cy5.5 (Biolegend), anti-CD14 and anti-CD19 FITC (both eBioscience), ant-CD45RA AF700 (BD Biosciences), anti-CXCR3 APC (BD Biosciences), anti-CD38 APC-Cy7 (Biolegend), and SYTOX Green (ThermoFisher) as a viability indicator for 15 minutes at 4°C and analyzed on an LSR II (BD Biosciences), gating on Viable CD4⁺ cells and excluding events that were positive for more than one tetramer color. Frequencies of tetramer positive cells were calculated as previously described (12 (link)).

James E.A., Gillette L., Durinovic-Bello I., Speake C., Bondinas G.P., Moustakas A.K., Greenbaum C.J., Papadopoulos G.K, & Kwok W.W. (2018). DRB4 has a distinct motif and presents a proinsulin epitope that is recognized in subjects with type 1 diabetes. Journal of immunology (Baltimore, Md. : 1950), 201(12), 3524-3533.

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Multiparametric Flow Cytometry of Liver Immune Cells

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Flow cytometric analysis was performed on CytoFLEX (Beckman Coulter, CA, USA). To analyze liver infiltrating cells, hematopoietic cells were isolated by MACS Liver dissociation kit (Miltnyi Biotec Inc.) and Gentle MACS (Miltnyi Biotec Inc.). Antibodies were sourced from Biolegend: anti-CD3 phycoerythrin/Cyanine7 (PE/Cy7; 17A2), anti-CD4 phycoerythrin (PE; GK1.5), anti-B220 PE (RA3-6B2), anti-CD90.2 PE (30-H12), anti-CD11c PE/Cy7 (N418), anti-F4/80 fluorescein isothiocyanate (FITC; BM8), anti-NK1.1 Alexa Fluor 488 (PK136), anti-CD49b PE (DX5), anti-CD44 allophycocyanin (APC; IM7), anti-CD62L allophycocyanin/Cyanine7 (APC/Cy7 (MEL-14), anti-CD25 PE (PC61.5), anti-CD69 FITC (H1.2F3), anti-CD49d PE (MFR4.B), anti-CXCR3 APC (CXCR3-173), anti-PD-1 PE (29F.1A12), anti-KLRG-1 APC (2F1/KLRG1), anti-LFA-1 PE (H155-78), anti-LPAM-1 PE (DATK32), anti-H-2 Kb APC (AF6-88.5) and anti-CD8a FITC (53–6.7), anti-CD11b PE (M1/70) from BD Biosciences.

Tamura S., Ishiguro H., Suwabe T., Katagiri T., Cho K., Fuse K., Shibasaki Y., Mikami T., Shindo T., Kitagawa H., Igarashi M., Sone H., Masuko M, & Ushiki T. (2023). Genetic manipulation resulting in decreased donor chondroitin sulfate synthesis mitigates hepatic GVHD via suppression of T cell activity. Scientific Reports, 13, 13098.

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Multi-marker Phenotypic Analysis of Peripheral Blood Leukocytes

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EDTA anticoagulated peripheral blood was used for phenotype analysis by flow cytometry. In detail, 200 μL of whole blood was lysed in 2 mL of VersaLyse at room temperature for 15 min. Then, cells were washed twice with FACS buffer, followed by resuspension in the appropriate antibody preparation and incubated for 20 min at 4°C. For intracellular staining of Ki-67 expression, we used permeabilization and fixation method using eBioscience perm/fix kit (ebioscience cat no. 88-8824-00). The following monoclonal antihuman antibodies were used in appropriate concentrations to stain cells: anti-CD45-Krome orange (Beckman Coulter, cat no. 96416), anti-CD14-PC5.5 (Beckman Coulter, cat no. A70204), anti-CD 19-APC-Alexa fluor 750 (Beckman Coulter, cat no.A94681), anti-CD27-PE (BD Pharmingen, cat no.555441), anti-CXCR3-APC (BD Pharmingen, cat no. 561732), anti-CXCR4-APC (BD Pharmingen, cat no. 560936), anti-CD95-APC (BD Pharmingen, cat no. 558814), anti-ki-67-PE (Ebioscience Cat no. 12-5699-42), and anti- IgD-FITC (BD Pharmingen, cat no. 555778). After staining, the cells were analyzed by 10-color flow cytometry (Navios, Beckman Coulter), with at least 20,000 CD19+ events collected for each analysis.

Mahmood Z., Schmalzing M., Dörner T., Tony H.P, & Muhammad K. (2020). Therapeutic Cytokine Inhibition Modulates Activation and Homing Receptors of Peripheral Memory B Cell Subsets in Rheumatoid Arthritis Patients. Frontiers in Immunology, 11, 572475.

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Profiling T-cell Subsets and Kv1.3 Channels

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We first isolated peripheral blood mononuclear cells (PBMCs) from peripheral venous blood by density gradient centrifugation. PBMCs were incubated in a modified RPMI medium (calcium concentration set to 2 mM) with conjugated anti-human monoclonal antibodies in order to differentiate T-lymphocyte subsets. We applied the following anti-human monoclonal antibodies: anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CXCR3 APC, and anti- CCR4 PE (all from BD Biosciences, San Diego, CA, USA). A portion of PBMCs was set apart for measurement of Kv1.3 channel expression, and these samples were additionally incubated with anti-Kv1.3 FITC (Sigma-Aldrich, St. Louis, MO, USA). The remainder of PBMCs were loaded with the calcium-sensitive fluorescent dyes Fluo-3 and Fura-Red (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. We then divided the PBMCs into 3 equal aliquots. The first aliquot was used as a control. In the second aliquot, Kv1.3 channels were blocked with margatoxin (MGTX, 60 nM), while in the third aliquot, IKCa1 channels were blocked with triarylmethane (TRAM, 60 nM). Samples were run on a Partec CyFlow ML flow cytometer (Munster, Germany). PBMCs were activated by the addition of 20 µg phytohemagglutinin (PHA), after the acquisition of a baseline for 2 min. Kinetic cell fluorescence data were recorded for another 10 min following activation.

Toldi G., Legány N., Ocsovszki I, & Balog A. (2020). Calcium Influx Kinetics and the Characteristics of Potassium Channels in Peripheral T Lymphocytes in Systemic Sclerosis. Pathobiology : journal of immunopathology, molecular and cellular biology, 87(5).

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Measuring Th1/Th2 Cell Calcium Dynamics

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Measurements were taken as described earlier [3] . Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by a standard density gradient centrifugation from 9 mL of freshly drawn peripheral venous blood and afterward kept in RPMI medium supplemented with CaCl2 (calcium concentration: 2 mM) throughout the following steps of the procedure. PBMCs were then incubated with the following conjugated anti-human monoclonal antibodies: anti-CD4 PE-Cy7, anti-CD8 APC-Cy7, anti-CXCR3 APC (for the determination of Th1 cells) and anti-CCR4 PE (for the determination of Th2 cells) (all from PharMingen, San Diego, CA, USA) according to the manufacturer's instructions. For monitoring cytoplasmic calcium levels, PBMCs were loaded with calcium-sensitive Fluo-3 and Fura Red dyes according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA, USA). PBMCs were incubated in the presence or absence of 4 nM MGTX or 240 nM TRAM. After recording a baseline for 2 min, cells were activated with 20 pg phytohemagglutinin (PHA) and the measurement was continued directly afterward in a kinetic manner for 15 min employing a flow cytometer (Beckman Coulter Gallios™, Miami, FL, USA). Recordings were evaluated with specific software (FacsKin, Budapest, Hungary) which is based on the calculation of a logistic function for each measurement [4] .

Toldi G., Munoz L., Herrmann M., Schett G, & Balog A. (2016). The effects of Kv1.3 and IKCa1 channel inhibition on cytokine production and calcium influx of T lymphocytes in rheumatoid arthritis and ankylosing spondylitis. Immunologic research, 64(2).

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Isolation and Characterization of Th1/2/17 Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples using Ficoll-Hypaque (GE Healthcare, USA) gradient centrifugation. To obtain Th1/2/17 cells, PBMCs were incubated for 30 min in with specific antibodies (FITC-anti-CD45RA, PE-anti-CCR6, Percep-anti-CD3, PE/Cy7-anti-CD4, APC-anti-CXCR3, BV421-anti-CXCR5, BV510-anti-CCR4; all from BD Biosciences) in PBS containing 2% fetal bovine serum. Cells were examined immediately using a BD FACS-CantoII, and data were analyzed using FlowJo software (TreeStar, USA). Cells were classified as follows: Th17 cells, CD3⁺CD4⁺CD45RA^-CXCR5^-CCR6⁺CCR4⁺; Th2 cells, CD3⁺CD4⁺CD45RA^-CXCR5^-CCR6^-CCR4⁺CXCR3^-; and TH1 cells, CD3⁺CD4⁺CD45RA^-CXCR5^-CCR6^-CCR4^-CXCR3⁺.

Yan S., Wu X., Jiang J., Yu S., Fang X., Yang H., Bai X., Wang H, & Luo X. (2022). Dupilumab improves clinical symptoms in children with Netherton syndrome by suppressing Th2-mediated inflammation. Frontiers in Immunology, 13, 1054422.

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Multi-Dimensional Immune Cell Profiling

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Single-cell suspensions were stained for 30 min using the following fluorophore-conjugated antibodies: FITC anti-CD3 (BD Biosciences Cat# 555332, RRID:AB_395739), APC/Cy7 anti-CD4 (BD Biosciences Cat# 341095, RRID:AB_400218), Percp-Cy5.5 anti-CXCR5 (BD Biosciences Cat# 562781, RRID:AB_2313576), PE anti-PD-1 (BD Biosciences Cat# 560795, RRID:AB_2033989), PE/Cy7 anti-CCR6 (BD Biosciences Cat# 560620, RRID:AB_1727440), APC anti-CCR6 (BD Biosciences Cat# 560619, RRID:AB_1727439), PECy7 anti-CXCR3 (BD Biosciences Cat# 560831, RRID:AB_2033944), APC anti-CXCR3 (BD Biosciences Cat# 565223, RRID:AB_2687488), FITC anti-CD19 (BD Biosciences Cat# 555412, RRID:AB_395812), BV421 anti-CD27 (BioLegend Cat# 302823, RRID:AB_10900425), APC anti-CD38 (BD Biosciences Cat# 555462, RRID:AB_398599), PECy7 anti-IgD (BD Biosciences Cat# 555776, RRID:AB_396111), and PE anti-CD138 (BD Biosciences Cat# 552026, RRID:AB_394323). For intracellular staining, cells were permeabilized using the Foxp3 Staining Buffer Set (eBioscience), then stained with PECy7 anti-Ki67 (BD Biosciences Cat# 561283, Clone B56), PE anti-Foxp3 (BD Biosciences Cat# 560082, RRID:AB_1645509), and PE anti-Bcl2 (BD Biosciences Cat# 340576, RRID:AB_400061). All samples were analyzed with an LSRFortessa flow cytometer (Beckton Dickinson), and data were processed with FlowJo software, version 10 (TreeStar, Inc.).

Wang Y., Liu Z., Wu J., Li F., Li G, & Dong N. (2020). Profiling circulating T follicular helper cells and their effects on B cells in post-cardiac transplant recipients. Annals of Translational Medicine, 8(21), 1369.

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