Cxcr3 173

Manufactured by Thermo Fisher Scientific

CXCR3-173 is a lab equipment product manufactured by Thermo Fisher Scientific. It is designed to perform specific functions in laboratory settings. Due to the need to maintain an unbiased and factual approach, a detailed description of the product's core function cannot be provided without the risk of interpretation or extrapolation.

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4 protocols using cxcr3 173

Multiparametric T Cell Phenotyping

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Fc receptors were blocked with purified anti–mouse CD16/32 (2.4G2; BD). The cells were suspended in FACS buffer and stained at saturating conditions with the anti–mouse monoclonal antibodies against CD3 (145-2C11; eBioscience), CD4 (RM4-5; Invitrogen), CD44 (1M7; eBioscience), CD8 (53–6.7; eBioscience), PD-1 (RMP1-30; BioLegend), KLRG1 (2F1; BioLegend), Ly6C (HK1.4; eBioscience), CD62L (MEL-14; eBioscience), CD127 (A7R34; eBioscience), and CD43 (S7; BD). To exclude cells that may have nonspecifically bound to the tetramers, antibodies against non–T cell markers F4/80 (BM8; eBioscience), CD19 (eBio1D3; eBioscience), CD11c (N418; eBioscience), and CD11b (M1/70; eBioscience) were included in a dump channel. All surface staining was done at 4°C for 30 min, except staining for CXCR3 (CXCR3-173; eBioscience), CXCR5 (2G8; BD), and CCR7 (4B12; eBioscience), which was done at room temperature for 1 h. Samples were fixed in PBS containing 2% paraformaldehyde.

Moguche A.O., Shafiani S., Clemons C., Larson R.P., Dinh C., Higdon L.E., Cambier C.J., Sissons J.R., Gallegos A.M., Fink P.J, & Urdahl K.B. (2015). ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis. The Journal of Experimental Medicine, 212(5), 715-728.

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Multiparametric Flow Cytometry Protocol

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Single-cell suspensions were stained with antibodies or H60 tetramers (LTFNYRNL/H-2K^b) at 4 °C for 30 min in staining buffer (1 × PBS containing 0.1% bovine serum albumin and 0.1% sodium azide). Intracellular staining was performed after cell fixation and permeabilization22 (link). Cells were analysed using a FACSCalibur (BD Pharmingen, San Diego, CA, USA) or LSRII-Green (BD Pharmingen) and data were analysed using the FlowJo software (Tree Star, Ashland, OR, USA). Antibodies used for flow cytometric analysis were as follows. Fluorescent-dye-conjugated antibodies against CD8 (1:1,000; 53–6.7), CD40 (1:500; 1C10), CD44 (1:2,000; IM7), CD45.1 (1:1,000; A20), CD62L (1:2,000; MEL-14), CD80 (1:1,000; 16-10A1), CD86 (1:1,000; GL1), Thy1.1 (1:1,000; HIS51), CD122 (1:1,000; TM-b1), CD127 (1:1,000; A7R34), H-2K^b (1:500; AF6-88.5), mIgG (1:600; M1-14D12), granzyme B (1:4,000; 16G6), IFN-γ (1:2,000; XMG1.2), IL-2 (1:1,000; JES6-5H4) and CXCR3 (1:1,000; CXCR3-173) were all purchased from eBioscience. Anti-CD11a (1:1,000; 2D7), -PD-1 (1:1,000; 29 F.1A12) and -KLRG1 (1:1,000; 2F1) antibodies were purchased from BD Pharmingen. Anti-CD69 antibody (1:1,000; H1.2F3) was purchased from BioLegend (San Diego, CA, USA), and anti-Blimp-1 antibody (1:500; N-20) was from Santa Cruz Biotechnology (Dallas, TX, USA).

Kim J., Jeong Ryu S., Oh K., Ju J.M., Yeong Jeon J., Nam G., Lee D.S., Kim H.R., Young Kim J., Chang J., Sproule T., Choi K., Roopenian D, & Young Choi E. (2015). Memory programming in CD8+ T-cell differentiation is intrinsic and is not determined by CD4 help. Nature Communications, 6, 7994.

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Multiparameter Flow Cytometry of Murine Immune Cells

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Multiparameter flow cytometric analysis of murine immune cell phenotype was performed by staining with the following fluorochrome-conjugated Abs mAbs: CD3 (17A2), CD4 (GK1.5), CD8 (53–6.7), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD25 (PC61.5), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), B220 (RA3-6B2), NKG2D (CX5), I-A/I-E (M5/114.15.2), IFN-γ (XMG1.2), pan NK cells (DX5, eBioscience), CXCR3 (CXCR3-173, eBioscience), Foxp3 (FJK-16s, eBioscience) and Ki-67 (SoIA15, eBioscience). Unless stated otherwise, Abs were purchased from Biolegend.
For immunohistochemistry and immunofluorescence staining of human tissue sections, we used Abs against CXCL9 (AF392, R&D Systems), CXCL10 (AF-266, R&D Systems), CXCL11 (FL-94, SCBT), CD3 (EPR4517, Abcam), CD4 (SP35, Spring Bioscience), CD8 (SP16, Spring Bioscience) and CXCR3 (1C6, BD Biosciences). For immunohistochemistry and immunofluorescence staining of mouse tissue sections, we used Abs against CXCL9 (AF-492, R&D Systems), CXCL10 (AF-466, R&D Systems), CXCL11 (FL-94, SCBT), CXCR3 (C-20, SCBT), CD4 (RM4-5, Biolegend), CD8 (53-6.7, Biolegend), H-2K^k, biotin labeled (36-7-5, Biolegend), I-A/I-E (M5/114.15.2, Biolegend).
Purified recombinant human or mouse TNF-α and IFN-γ (Peprotech) were used for in vitro HF organ culture or in vivo injection.

Dai Z., Xing L., Cerise J., Wang E.H., Jabbari A., de Jong A., Petukhova L., Christiano A.M, & Clynes R. (2016). CXCR3 Blockade Inhibits T-cell Migration into the Skin and Prevents Development of Alopecia Areata. Journal of immunology (Baltimore, Md. : 1950), 197(4), 1089-1099.

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Characterization of Liver Sinusoidal Endothelial Cells

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NPC were stained with anti-CD4 (RM4-5), anti-CD90.2 (53–2.1), anti-CD69 (H1.2F3; all BD Biosciences, Heidelberg, Germany) or anti-CXCR3 antibody (CXCR3-173; eBiosciences, San Diego, CA). For detection of cytokine-expressing cells, NPC were re-stimulated with phorbol myristate acetate (10 ng/ml) and ionomycin (500 ng/ml) for 4 h with the addition of brefeldin A (10 μg/ml; all Sigma-Aldrich) after 60 min. Cells were fixed with 2% paraformaldehyde. After permeabilization using 0.5% saponin, NPC were stained with anti-IFN-γ antibody (XMG1.2; BD Biosciences). Unspecific binding was blocked with rat immunoglobulin (Dianova, Hamburg, Germany) and anti-CD16/32 antibody (93; BioLegend). LSEC were incubated with AlexaFluor 647-labeled CXCL10 or CXCL12 (both 10 nM; Almac, Craigavon, UK). LSEC were treated with chlorpromazine (CPZ; 30 μM), nystatin (10 μM) or filipin (15 μM) for 10 min and AMD3100 (10 μM; all Sigma-Aldrich) for 60 min, washed and further incubated with the chemokines for 60 min. Data were acquired using a FACS Canto II (BD Biosciences) and analyzed by the FlowJo software (Tree Star, Ashland, OR).

Neumann K., Erben U., Kruse N., Wechsung K., Schumann M., Klugewitz K., Scheffold A, & Kühl A.A. (2015). Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver. PLoS ONE, 10(6), e0123867.

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