Cells were resuspended in FACS buffer (Dulbecco’s PBS + 1% bovine serum albumin). Non-specific staining was blocked with mouse serum and anti-mouse CD16/CD32 (clone 2.4G2, BD Biosciences, San Jose, CA, USA) for 15 min on ice. Cells were stained for 30 min on ice with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP), or allophycocyamin (APC) conjugated mAb (all for BD Biosciences expect when indicated) specific for CD45 (clone Ly-5), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD11b (M1/70), CD25 (clone PC61), MHC class II (clone 2G9), CD44 (clone IM7), CD62L (clone MEL-14), CD69 (clone H1-2F3), and CXCR3 (R&D Systems, Minneapolis, MN, USA). Virus-specific CD8 T cells were characterized using H-2Db/S510 MHC class I tetramers (27 (link)). Intracellular staining for Foxp3 (eBioscience, San Diego, CA, USA) was performed according to the manufacturer’s instructions. Cells were washed twice with FACS buffer and analyzed using a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (Tree Star Inc., Ashland, OR, USA).
Cxcr3
Manufactured by R&D Systems
Sourced in United States, United Kingdom
CXCR3 is a cell surface receptor protein that binds to chemokines. It is involved in the regulation of immune cell trafficking and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Cxcr1, by R&D Systems (3 mentions)
Ventana optiview dab kit, by Roche (3 mentions)
Cxcl10, by R&D Systems (3 mentions)
Cxcr4, by R&D Systems (2 mentions)
Cxcr3b, by Creative Biomart (2 mentions)
β actin, by Merck Group (2 mentions)
Cxcl13, by R&D Systems (2 mentions)
Benchmark xt, by Roche (2 mentions)
Anti mouse cd16 cd32 clone 2.4g2, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Ccl27, by Thermo Fisher Scientific (1 mentions)
Cxcr5, by R&D Systems (1 mentions)
Cxcl10, by Thermo Fisher Scientific (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
P erk, by Merck Group (1 mentions)
S100a8 a9, by Abcam (1 mentions)
Ifn γ, by BD (1 mentions)
Interleukin 4 (il 4), by BD (1 mentions)
Anti il 17 mab, by Thermo Fisher Scientific (1 mentions)
Collagenase a, by Merck Group (1 mentions)
13 protocols using cxcr3
1
Comprehensive Immune Cell Phenotyping by Flow Cytometry
2
Chemokine Receptor Flow Cytometry
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FITC-conjugated mouse antihuman CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR1, CXCR3, CXCR4, and CXCR5 or unconjugated monoclonal mouse-antihuman CCR1, CCR2, and CXCR6, as well as PE-conjugated rat antihuman CCR8, PE-conjugated rat antihuman CCR10, and PE-conjugated rat IgG2b, were obtained from R&D Systems Europe Ltd. (Abingdon, UK). FITC-conjugated mouse antihuman CX3CR1 was purchased from Medical and Biological Laboratories Co. Ltd. (Nagoya, Japan). FITC-conjugated monoclonal mouse antihuman CD3, PE-conjugated monoclonal mouse antihuman CD56, and FITC-conjugated goat anti-mouse were purchased from Becton-Dickinson (San Diego, CA, USA). FITC-conjugated mouse IgG, PE-conjugated mouse IgG, unconjugated mouse IgG, and unconjugated rat IgG were obtained from either Becton-Dickinson or from R&D Systems. Pertussis toxin (PTX), MMF, and DMF were obtained from Sigma-Aldrich (Saint Louis, MO, USA). CCL1, CCL27, CCL28, and CXCL10 were purchased from PeproTech (London, UK).
3
Protein Expression Analysis in Cells
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Protein extracts were analyzed by standard methods and antibodies to CXCR3
(R&D Systems, Minneapolis, MN, USA); CXCR3B (Creative BioMart, Shirley, NY,
USA); ERK (Cell Signaling Technology, Danvers, MA, USA); p-ERK (Sigma-Aldrich,
St. Louis, MO, USA); CREB, p-CREB, NOTCH 1 (Cell Signaling Technology); STAT3,
p-STAT3, and β-actin (Sigma-Aldrich). Densitometry was performed using ImageJ
software.
(R&D Systems, Minneapolis, MN, USA); CXCR3B (Creative BioMart, Shirley, NY,
USA); ERK (Cell Signaling Technology, Danvers, MA, USA); p-ERK (Sigma-Aldrich,
St. Louis, MO, USA); CREB, p-CREB, NOTCH 1 (Cell Signaling Technology); STAT3,
p-STAT3, and β-actin (Sigma-Aldrich). Densitometry was performed using ImageJ
software.
4
Immunohistochemical Profiling of Inflammatory Markers
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Immunohistochemistry were performed on the formalin-fixed paraffin-embedded sections using a Benchmark XT automated staining system (Ventana Medical Systems Inc., Tucson, AZ, USA). The primary antibodies used were: CD4, 1:10 and CD8, 1:50 (Thermo Fisher Scientific, Fremont, CA, USA); CD68, 1:200 (Novocastra Laboratories Ltd, Newcastle, UK); CXCL10, 1:50; CXCL13, 1:50; and CXCR3, 1:20 (R & D Systems). Detection was achieved using a Ventana Optiview DAB kit (Ventana Medical Systems). Scores were calculated by dividing the numbers of positive inflammatory cells by the numbers of all inflammatory cells, expressed as percentages (CXCL10, CXCL13, and CXCR3) or graded on a scale from 1 to 3: 1:1–33 %; 2, 34–66 %; and 3, 67–100 % (CD4, CD8, and CD68).
5
Immunohistochemical Profiling of Immune Markers
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Formalin-fixed, paraffin-embedded sections were analyzed by IHC using a Benchmark XT automated staining system (Ventana Medical Systems Inc., Tucson, AZ, USA). The primary antibodies used were [anti- (dilution factor; manufacturer)]: CD4 (prediluted; Roche, Basel, Switzerland), CD8 (prediluted; Roche), CD21 (prediluted; Roche), CXCL10 (1:30; R & D Systems, Minneapolis, MN, USA), CXCL13 (1:50; R & D Systems), CXCR3 (1:20; R & D Systems), S100A8/A9 (1:900; Abcam, Cambridge, UK), and programmed death-1 (PD-1, 1:50, Cell Marque, Rocklin, CA, USA). Staining was detected using a Ventana Optiview DAB kit (Ventana Medical Systems Inc.). The results for CXCL10, CXCL13, CXCR3 and S100A8/A9 IHC were graded from 1 to 3 according to the percentage of positive lymphoid cells and histiocytes (1: 1–33%, 2: 34–66%, 3: 67–100%).
6
Multiparametric T-cell immunophenotyping
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The medium used was RPMI 1640 (Seromed) supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 2x10-5 M 2-mercaptoethanol (2-ME; all from Invitrogen), and 10% fetal cow serum. FITC-, PE-, allophycocyanin (APC), peridin chlorophyll protein (PerCP), Pacific Blue – conjugated anti-CD3, CD4, CD8, CD161, -IFN-gamma, -IL-4, -TNF-α were from BD Bioscience. Anti – IL-17 mAb was obtained from eBioscience, and anti- CRTH2, -CCR6 were from Miltenyi, CXCR3 was from R&D Systems. PMA, ionomycin, brefeldin A and Collagenase A were purchased from Sigma-Aldrich. rIL-2 (Proleukin®) from Novartis, the polyclonal activator phytohemagglutinin (PHA) from Biochrom AG, and the recall antigen streptokinase (SK) from CSL Behring; rh TG2 and TG3 were purchased from Zedira.
7
Phenotypic Characterization of GMP-ASC
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GMP‐ASC alone and treated either with OA‐CM or OA‐SF were characterized by flow cytometry using the following markers CXCR1, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR5 (R&D), CXCR7 (Abcam, Cambridge, UK), and IL6R (GeneTex Inc., Irvine, CA).
In brief, after harvesting cells upon detachment, they were washed twice with phosphate‐buffered saline (PBS), centrifuged, and washed in a flow cytometry buffer (PBS supplemented with 2% bovine serum albumin [BSA] and 0.1% sodium azide).
Aliquots of 1 × 105 cells were then incubated with primary antibodies at 10 µg/ml 4 °C for 30 min, washed twice with a flow cytometry buffer, and incubated with polyclonal rabbit anti‐mouse and goat anti‐rabbit immunoglobulins/fluorescein isothiocyanate (FITC) conjugate (Dako Cytomation, Glostrup, Denmark) at 4 °C for 30 min. After two final washes, the cells were analyzed using a fluorescence‐activated cell sorting (FACS) CantoII Cytometer (Becton Dickinson). For isotype control, non‐specific mouse IgG was substituted for the primary antibody.
In brief, after harvesting cells upon detachment, they were washed twice with phosphate‐buffered saline (PBS), centrifuged, and washed in a flow cytometry buffer (PBS supplemented with 2% bovine serum albumin [BSA] and 0.1% sodium azide).
Aliquots of 1 × 105 cells were then incubated with primary antibodies at 10 µg/ml 4 °C for 30 min, washed twice with a flow cytometry buffer, and incubated with polyclonal rabbit anti‐mouse and goat anti‐rabbit immunoglobulins/fluorescein isothiocyanate (FITC) conjugate (Dako Cytomation, Glostrup, Denmark) at 4 °C for 30 min. After two final washes, the cells were analyzed using a fluorescence‐activated cell sorting (FACS) CantoII Cytometer (Becton Dickinson). For isotype control, non‐specific mouse IgG was substituted for the primary antibody.
8
Phenotype Analysis of Expanded Cells
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The phenotype of expanded cells and PBMCs at baseline (day 0), before the 3rd administration (day 14), and 4 weeks after 3rd administration (day 42) were analyzed by flow cytometry. Monoclonal antibodies specific for CD3, CD16, CD56 (Beckman Coulter, CA, USA), NKG2D (eBioscience, CA, USA), CXCR3 (R&D system, MN, USA), CXCR4 (Becton–Dickinson, CA, USA), CX3CR1 (Bio Legend, CA, USA) were applied. Each monoclonal antibody was conjugated as follows: CD3 CD16, CD69, CXCR3 with fluorescein isothiocyanate (FITC)-, CD56, NKG2D, CXCR4, CX3CR1 with phycoerythrin (PE)-, CD3 with phycoerythrin-Cyanin 5 (PE-Cy5)-, CD56 with phycoerythrin-Cyanin 7 (PC7-Cy7)-. Cells were analyzed by Cytomics FC500 (Beckman Coulter) and data were acquired by the CXP software, version 2.2 (Beckman Coulter) according to the manufacturer’s instruction.
9
Immunohistochemical Analysis of CXCR2 and CXCR3
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Tissue microarray (TMA) slides were bought from Biomax (US Biomax Inc., Rockville, MD, USA). After deparaffinizing, rehydrating, heat‐induced epitope retrieval and blocking with 3% H2O2, the treated TMA slides were incubated with CXCR2 and CXCR3 (R&D Systems) primary antibody and the immunohistochemistry was performed as described previously 23 . The slides were analysed using the Aperio ImageScope (Aperio Technologies Inc., Vista, CA, USA) and a digital stained cell score was obtained. The detail clinicopathologic characteristics of the patients included in the TMA are listed in Table S2.
10
Multiparametric Flow Cytometry Analysis
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