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Cxcr3 fitc

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CXCR3 - FITC is a fluorescently labeled antibody that specifically binds to the CXCR3 chemokine receptor. CXCR3 is a G protein-coupled receptor that plays a role in the trafficking and recruitment of T cells to sites of inflammation.

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Lab products found in correlation

Cd3 bv605, by BioLegend (3 mentions) Cd4 apc efluor 780, by Thermo Fisher Scientific (3 mentions) Cd8 bv650, by Thermo Fisher Scientific (3 mentions) Pd 1 apc, by Thermo Fisher Scientific (2 mentions) Facsdiva software, by BD (2 mentions) Cd25 pe, by Thermo Fisher Scientific (2 mentions) Ccr7 pe, by BioLegend (2 mentions) Lsrfortessa, by BD (2 mentions) Histopaque 1077, by Merck Group (2 mentions) Igd fitc, by BD (2 mentions) Cd27 pe cy7, by Thermo Fisher Scientific (2 mentions) Cd127 apc, by Thermo Fisher Scientific (2 mentions) Cd45ra percp cy5, by Thermo Fisher Scientific (2 mentions) Cd19 bv450, by BD (2 mentions) Cd62l apc ef780, by Thermo Fisher Scientific (2 mentions) Cxcr5 af488, by BioLegend (2 mentions) Hla dr efluor450, by Thermo Fisher Scientific (2 mentions) Cd8 pe cy7, by BioLegend (1 mentions) Cell staining buffer, by BioLegend (1 mentions) Cd45ra apc vio770, by Miltenyi Biotec (1 mentions)

Close spelling variants of this product

Anti-CXCR3 FITC (6 citations)

7 protocols using cxcr3 fitc

1

Characterization of CAR-T Cells by Flow Cytometry

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The following antibodies were used: CD3 (VioBlue; Miltenyi Biotech or Pacific blue and PE; BioLegend), CD4 (FITC or APC-Cy7; BioLegend), CD8 (PE-Cy7; BioLegend), CD3/CD19 antibodies (FITC/PE; BD), CD28 (PerCP-Cy5.5; eBioscience), PD-1 (FITC; clone: EH12.2H7; BioLegend), TIM-3 (APC-Cy7; BioLegend), LAG-3 (VioBlue; Miltenyi Biotech), CD45RA (APC-Vio770; Miltenyi Biotec or Brilliant Violet; BioLegend), CCR7 (PerCP-Vio770; Miltenyi Biotec or PerCP; BioLegend), CCR2 (APC; Biolegend), CCR4 (PE; Biolegend), CCR5 (Alexa Influenza 488; Biolegend), CXCR2 (PE-Cy7; Biolegend) and CXCR3 (FITC; Biolegend).
Transduction efficacy was determined on day 6 and day 9 of culture by labeling CAR-T cells with biotin-labeled polyclonal goat anti-mouse F(ab)2 antibody (anti-Fab, Jackson Immunoresearch, West Grove, Pennsylvania) and streptavidin (APC conjugated; BioLegend). CD3+F(ab)2+ cells were defined as CAR-T cells. Isotype labeled cells (Jackson Immunoresearch) and untransduced cells served as negative controls. For further characterization, cells were stained with antibodies mentioned above. Cells were washed and re-suspended in cell staining buffer (BioLegend), incubated for 30 min with the antibodies on ice, washed and measured using MACSQuant FACS cytometer (Miltenyi Biotec). Samples were analyzed by FlowJo software (FlowJo LLC, Ashland, Oregon).
Itzhaki O., Jacoby E., Nissani A., Levi M., Nagler A., Kubi A., Brezinger K., Brayer H., Zeltzer L.A., Rozenbaum M., Vernitsky H., Markel G., Toren A., Avigdor A., Schachter J, & Besser M.J. (2020). Head-to-head comparison of in-house produced CD19 CAR-T cell in ALL and NHL patients. Journal for Immunotherapy of Cancer, 8(1), e000148.
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2

Comprehensive Immunophenotyping of T Cell Subsets

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Blood was collected and processed within 4 hours maximum. An ammonium chloride-based lysing reagent (BD Pharm Lyse, BD Biosciences) was used for erythrocyte deletion of 1ml of blood. After washing, cells were suspended in PBS and stained with Aqua Dye (Invitrogen) for cell viability. Cells were washed again, suspended in staining buffer and divided in four tubes. The four different panels assessed contained some common and some specific antibodies. Common antibodies were: CD3-eFluor 605, CD4-Alexa700 (eBioscience, San Diego, CA), CCR7-Horizon PE-CF594, CD38-Brillant Violet 421, HLA-DR-PerCP-Cy5.5 and CD11c-PE-Cy7 (BD Biosciences). Specific for each panel were: 1) CCR2-PE, CCR5-APC-Cy7, CXCR6-APC (R&D Systems Inc.) and CXCR3-FITC (BioLegend); 2) CD49d (α4)-FITC, β7-APC, CCR9-PE (BD Biosciences) and CD29 (β1)–APC-Cy7 (BioLegend); 3) CD103-FITC, CD54-APC, CD49a (α1)-PE and CD29–APC-Cy7 (BioLegend); 4) CD18-APC, CLA-FITC (BD Biosciences) and CCR10-PE (BioLegend). Cells were acquired using a BD LSRFortessa SORP flow cytometer (Flow Cytometry Platform, IGTP) and analyzed with FlowJo 9.3.2 software (TreeStar). Gates were drawn based on fluorescence minus one-controls and isotypes, and CD3+ CD4- phenotype was considered CD8+ T cells.
Qualai J., Cantero J., Li L.X., Carrascosa J.M., Cabré E., Dern O., Sumoy L., Requena G., McSorley S.J, & Genescà M. (2016). Adhesion Molecules Associated with Female Genital Tract Infection. PLoS ONE, 11(6), e0156605.
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3

Multiparametric Flow Cytometry Immunophenotyping

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Peripheral blood mononuclear cells were prepared for analysis by density centrifugation using Histopaque-1077 (Sigma-Aldrich). The following antibodies were used for flow cytometry immunophenotyping: CD3 – BV605 (Biolegend, San Diego, CA, USA), CD4 – APC-eFluor780 (eBioscience, San Diego, CA,USA), CD8 – BV650 (eBioscience, San Diego, CA,USA), CD25 – PE (eBioscience, San Diego, CA,USA), CD127 – APC (eBioscience, San Diego, CA,USA), CD45RA – PerCP-Cy5.5(eBioscience, San Diego, CA,USA, CD19 – BV450 (BD Bioscience, Franklin Lakes, NJ, USA), CD27 – PE-Cy7 (eBioscience, San Diego, CA,USA, CD62L – APC-eF780 (eBioscience, San Diego, CA,USA, CXCR3 – FITC (Biolegend, San Diego, CA, USA), CXCR5 – AF488 (Biolegend, San Diego, CA, USA), CCR7 – PE (Biolegend, San Diego, CA, USA), PD-1 – APC (eBioscience, San Diego, CA,USA), HLA-DR- eFluor450 (eBioscience, San Diego, CA, USA), IgD – FITC (BD Bioscience, Franklin Lakes, NJ, USA). Flow cytometry analysis was performed on a BD LSRFortessa (BD Bioscience) with FACS Diva software (BD Bioscience) for acquisition, then analysis was performed with FlowJo software (LLC).
Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.
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4

Quantifying Antigen-Specific T-Cell Frequencies

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Analysis of T-cell frequency was accomplished using our previously published approach (26 (link)). Briefly, 40–60 × 106 PBMCs were resuspended in a total of 400–600 µL media, divided into two or three independent tubes of 20 × 106 cells (200 µL) each, incubated with 50 nmol/L dasatinib for 10 min at 37°C, and stained with 20 µg/mL PE-labeled, PE-CF594–labeled, or PE-Cy5–labeled HIP tetramers at room temperature for 120 min (three tetramers per tube for a total of six hybrid peptide tetramers plus an nonimmunogenic control tetramer if adequate cells were available for a third staining tube). Cells were washed, incubated with PE-magnetic beads (Miltenyi Biotec) for 20 min at 4°C, and magnetically enriched; 1% of the cells were retained as a nonenriched sample. Enriched (bound) and nonenriched (precolumn) samples were stained with CD4 V500, CD14 PerCP-Cy5.5, and CD19 PerCP-Cy5.5 (eBioscience) and CD45RA AF700 (BD), CXCR3 FITC, CCR6 BV421, and CCR4 BV605 (BioLegend) for 15 min at 4°C. After washing, cells were labeled with ViaProbe (BD Biosciences) and analyzed on a FACSCanto (BD Biosciences), gating on CD4+CD14−CD19−ViaProbe− cells and plotting tetramer versus CD45RA. Frequencies were calculated as previously described (26 (link)).
Arribas-Layton D., Guyer P., Delong T., Dang M., Chow I.T., Speake C., Greenbaum C.J., Kwok W.W., Baker R.L., Haskins K, & James E.A. (2020). Hybrid Insulin Peptides Are Recognized by Human T Cells in the Context of DRB1*04:01. Diabetes, 69(7), 1492-1502.
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5

Multiparametric Flow Cytometry Immunophenotyping

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Peripheral blood mononuclear cells were prepared for analysis by density centrifugation using Histopaque-1077 (Sigma-Aldrich). The following antibodies were used for flow cytometry immunophenotyping: CD3 – BV605 (Biolegend, San Diego, CA, USA), CD4 – APC-eFluor780 (eBioscience, San Diego, CA,USA), CD8 – BV650 (eBioscience, San Diego, CA,USA), CD25 – PE (eBioscience, San Diego, CA,USA), CD127 – APC (eBioscience, San Diego, CA,USA), CD45RA – PerCP-Cy5.5(eBioscience, San Diego, CA,USA, CD19 – BV450 (BD Bioscience, Franklin Lakes, NJ, USA), CD27 – PE-Cy7 (eBioscience, San Diego, CA,USA, CD62L – APC-eF780 (eBioscience, San Diego, CA,USA, CXCR3 – FITC (Biolegend, San Diego, CA, USA), CXCR5 – AF488 (Biolegend, San Diego, CA, USA), CCR7 – PE (Biolegend, San Diego, CA, USA), PD-1 – APC (eBioscience, San Diego, CA,USA), HLA-DR- eFluor450 (eBioscience, San Diego, CA, USA), IgD – FITC (BD Bioscience, Franklin Lakes, NJ, USA). Flow cytometry analysis was performed on a BD LSRFortessa (BD Bioscience) with FACS Diva software (BD Bioscience) for acquisition, then analysis was performed with FlowJo software (LLC).
Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.
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6

Intracellular STAT1 Kinetics by FACS

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Intracellular pSTAT1 and total STAT1 kinetics were determined by FACS analysis. 50 µl whole blood was stimulated according to BD Phosflow™ T Cell Activation Kit Instruction Manual in polystyrene round-bottom tubes (Becton Dickinson Falcon). Blood was stimulated with Imukin® 100ng/ml for 0–180min at 37°C. Erythrocytes were lysed and leucocytes fixed with 1 ml of pre-warmed 1x lyse/fix buffer. The cells were then permeabilized by adding 300 µL of cold perm buffer III on ice for 30 min, stained with 750 µl stain buffer and incubated for 1 h in the dark at 4°C with following antibodies: anti-human CD14-FITC (BD Biosciences 345784), isotype STAT normal mouse IgG2a Alexa Fluor® 647 (Santa Cruz Biotechnology sc-24637) and Alexa Fluor® 647 mouse Anti-Stat1 (pY701) (BD Biosciences 612597). For Th1 and Th17 percentages PBMCs were stained with CD3 – BV605 (Biolegend), CD4 - APC-eFluor780 (eBioscience), CD8 – BV650 (eBioscience), CXCR3 – FITC (BioLegend), CCR6 – PE-Cy7 (BioLegend), CD45RA – PerCP (eBioscience) GZMB - e450 (BioLegend), CFSE - Invitrogen. All data were collected with LSR II (Becton Dickinson) and analyzed with FlowJo software (Treestar, Ashland, OR, USA).
Körholz J., Gabrielyan A., Sowerby J.M., Boschann F., Chen L.S., Paul D., Brandt D., Kleymann J., Kolditz M., Toepfner N., Knöfler R., Jacobsen E.M., Wolf C., Conrad K., Röber N., Lee-Kirsch M.A., Smith K.G., Mundlos S., Berner R., Dalpke A.H., Schuetz C, & Rae W. (2021). One Gene, Many Facets: Multiple Immune Pathway Dysregulation in SOCS1 Haploinsufficiency. Frontiers in Immunology, 12, 680334.
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7

Detailed Immunophenotyping Assay Protocol

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Chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO). Anti–mouse flurochrome labeled antibodies (Abs): CD4-PB, CD8-APC-Cy7, CD62L-APC, CD44-PerCP-Cy5.5, CD127-PE, KLRG1-PE, IFN-γ-PECy7, TNFα-PerCPCy5.5, IL-17-PerCPCy5.5 and Abs for ELISA were procured from BD Pharmingen (San Diego, CA); CD27-FITC, CD43-PE, CXCR3-FITC and CCR6-APC from Biolegend (San Diego, CA) or otherwise mentioned. RPMI-1640 and FBS were purchased from GIBCO (Grand Island, NY). For culturing of cells, tissue culture grade plastic-ware was purchased from BD Biosciences (Bedford, MA).
Rai P.K., Chodisetti S.B., Maurya S.K., Nadeem S., Zeng W., Janmeja A.K., Jackson D.C, & Agrewala J.N. (2018). A lipidated bi-epitope vaccine comprising of MHC-I and MHC-II binder peptides elicits protective CD4 T cell and CD8 T cell immunity against Mycobacterium tuberculosis. Journal of Translational Medicine, 16, 279.
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