LLC cells were obtained from ATCC and were cultured in complete DMEM with 10% FBS and penicillin/streptomycin. Splenic T cells were isolated from C57BL/6 mice and were stimulated in T cell medium (RPMI 1640, penicillin/streptomycin, 200 mM L-glutamine, 50 mM β-mercaptoethanol, 1 M HEPES) with or without L-arginine (75 μM) and nor-NOHA (Sigma-Aldrich). Purified anti–mouse CD3 and purified anti–mouse CD28 antibodies were purchased from BioLegend (catalog numbers 100339 and 102122, respectively). IFN-γ, ANXA2, and ARG1 ELISAs were performed using R&D Systems Quantikine ELISA kits. Recombinant ANXA2 (R&D Systems), IL-4, and IL-10 (Life Technologies), LTA (US Biological), and LPS (Sigma-Aldrich) were used at the indicated concentrations.
Nor noha
Manufactured by Merck Group
Sourced in Germany
Nor-NOHA is a laboratory reagent that functions as a potent and selective inhibitor of the enzyme arginase. It is utilized in research applications to study the role of arginase in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
L nmma, by Merck Group (6 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd3, by BioLegend (1 mentions)
L arginine, by Merck Group (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Sc 52893, by Santa Cruz Biotechnology (1 mentions)
Recombinant tgf β, by Santa Cruz Biotechnology (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (1 mentions)
Anti cd3 anti cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Clone rpa t4, by BD (1 mentions)
Anti cd8 apc cy7, by BioLegend (1 mentions)
Clone rpa t8, by BioLegend (1 mentions)
Nor noha, by Enzo Life Sciences (1 mentions)
Arginase activity assay kit, by Merck Group (1 mentions)
Anti ifnγ xmg1.2, by Thermo Fisher Scientific (1 mentions)
3h thymidine, by Cytiva (1 mentions)
Celltrace violet dye, by Thermo Fisher Scientific (1 mentions)
12 protocols using nor noha
1
Arginine Modulates T Cell Activation
2
Mechanisms of Treg Expansion by MDSCs
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To unravel the mechanism of the Treg expanding effect of MDSCs, CD4 T cells were cultured with MDSCs (at a 1:1 ratio) under the Th0 condition for 72 h in the presence or absence of recombinant TGF-β (10 μg/ml, #sc-52893; Santa Cruz Biotechnology, Inc.), l -NMMA (500 μM, Sigma-Aldrich, #M7033), an iNOS inhibitor, and nor-NOHA (500 μM, Sigma-Aldrich, #399275), an Arg1 inhibitor. After that, cells were stained with anti-FOXP3 PE using intracellular cytometric analysis.
3
Modulation of T cell proliferation by granulocytes
4
Inhibition of Arginase Activity in Asthmatic Mice
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Mice were injected i.n. with 200 µg of nor-NOHA (N-hydroxy-nor-l -arginine, Enzolifesciences) in 50 µl of PBS on days 8, 15, 18, 21, 24, and 27 post first mold instillation. In order to verify the effectiveness of the inhibitor, we measured the arginase activity using the Arginase Activity Assay kit (Sigma MAK112) on naive and asthmatic lung extracts in the presence or absence of nor-NOHA (2 mg/ml) during the arginase reaction.
5
Modulation of T-cell Responses by MDSCs
6
Tumor-Derived Myeloid Cell Suppression of T-Cell Proliferation
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2 Â 10 5 neutrophils or monocytes sorted from tumors were added to 2 Â 10 5 na€ ve C57BL/6 splenocytes stimulated with anti-CD3 (1 mg/mL, clone 145-2C11, BD Biosciences, cat# 550275) and anti-CD28 (2 mg/mL, clone 37.51, eBioscience, cat# 16-0281-85) and cultured in flat-bottom 96-well plates in culture medium for ex vivo cell culture described above. After 24 hours of culture at 37 C, 1 mCi (0.037 MBq) 3 H-thymidine (PerkinElmer, cat# NET027A005MC) was added, and after another 18 hours of culture, T-cell proliferation was measured as count per minute in a liquid scintillation counter.
For measuring T-cell proliferation via flow cytometry, splenocytes were labeled with CellTrace Violet dye (Thermo Fisher, cat# C34571) following the manufacturer's instructions and cocultured with tumorderived neutrophils as described above. To inhibit potential T-cellsuppressive pathways, 0.5 mmol/L Nw-Nitro-L-arginine methyl ester (L-NAME, Sigma, cat# N5751), 0.5 mmol/L Nw-Hydroxy-nor-Larginine (Nor-NOHA, Sigma, cat# 399275), or 200 U/mL superoxide dismutase (Sigma, cat# S5395) was added to the cocultures. After 42 hours of culture at 37 C, the frequency of CellTrace low proliferating T cells was determined via flow cytometry.
For measuring T-cell proliferation via flow cytometry, splenocytes were labeled with CellTrace Violet dye (Thermo Fisher, cat# C34571) following the manufacturer's instructions and cocultured with tumorderived neutrophils as described above. To inhibit potential T-cellsuppressive pathways, 0.5 mmol/L Nw-Nitro-L-arginine methyl ester (L-NAME, Sigma, cat# N5751), 0.5 mmol/L Nw-Hydroxy-nor-Larginine (Nor-NOHA, Sigma, cat# 399275), or 200 U/mL superoxide dismutase (Sigma, cat# S5395) was added to the cocultures. After 42 hours of culture at 37 C, the frequency of CellTrace low proliferating T cells was determined via flow cytometry.
7
Calcium-sensing receptor and ornithine decarboxylase regulation
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The calcium-sensing receptor protein was detected by the anticalcium sensing receptor antibody produced in rabbit (Sigma-Aldrich Chemie, Taufkirchen, Germany; SAB4503369M). Ornithine decarboxylase was detected by the anti-ODC antibody produced in goat (Santa-Cruz Biotechnology, Santa Cruz, CA, USA; F-14, sc-21516). The calcium-sensing receptor and ornithine decarboxylase levels were normalized to actin (rabbit antiactin antibody (Sigma-Aldrich Chemie, Taufkirchen, Germany; A2668)). Secondary antibodies were directed against rabbit IgG and coupled to alkaline phosphatase (Affinity Biologicals; Ancaster, ON, Canada; GAM-APHRP). SB202190, chelerythrine chloride, and SP600125 were purchased from Calbiochem (Merck Darmstadt, Darmstad, Germany). Uric acid, L-nitro arginine methyl ester (L-NAME), Nor-NOHA, and Difluoromethyl ornithine (DFMO) were purchased from Sigma-Aldrich Chemie, Taufkirchen, Germany.
8
Isolation and Activation of Naive CD4+ T Cells
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Naive CD4 T cells were pre-enriched from murine spleens by magnetic sorting using anti-CD4 microbeads (Miltenyi Biotec) labeling followed by positive selection using an autoMACS (Miltenyi Biotec), as described by the manufacturer. Pre-enrichment was followed by fluorescence-activated cell sorting (FACS) with the target cells having the phenotype of single CD90.2+CD4+CD62LhighCD44– CD25– events. 2 × 105 CFSE-labeled naive CD4+ T cells were incubated in cRPMI in 96-wells plates for 4 days at 37 °C, >95% humidity and 5% CO2 in the presence or absence of plate-bound 0.5 μg/ml anti-CD3 and 0.1 μg/ml anti-CD28 antibodies (145-2C11 and 37.51, both from eBioscience). CD3–CD19–CD49b–CD11b+Ly6C++Ly6G– cells from digested lungs of IAV-infected mice were FACS-sorted and added to the cultures. 500 μg/ml L-NMMA, 200 μM 1-MDT (both from Sigma-Aldrich), and 500 μg/ml NOR-NOHA (Merck) were used to block iNOS, IDO, and Arg1, respectively.
9
T-Cell Proliferation Assay with Inhibitors
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Mouse or human T cells were labeled with CellTrace Violet (Thermo Fisher Scientific), plated in 96‐well plates, and activated with mouse‐ or human‐specific CD3/CD28‐Dynabeads (Thermo Fisher Scientific). Mouse T‐cell medium consisted of RPMI1640 + GlutaMAX (GIBCO), 10% fetal bovine serum (GIBCO), 10 μg/ml Gentamycin (GIBCO), 10 mM HEPES (BioConcept), 1 mM Sodium Pyruvate (GIBCO), 1× MEM‐NEAA (GIBCO), and 50 μM beta‐mercaptoethanol (GIBCO). Human T‐cell medium consisted of RPMI1640 + GlutaMAX, 8% human serum (Biowest, France), and 10 μg/ml Gentamycin. Where specified, small molecule inhibitors (see below), neutralizing antibodies (Appendix Table S3 ), and myeloid cells (neutrophils, monocytes, or macrophages) were added within 1 h after plating of T cells. Small molecule inhibitors were Acetylsalicylic acid (Sigma), Adenosine receptor 2A inhibitor (SCH58261; Cayman Chemicals), Galunisertib (LY2157299; Cayman Chemicals), L‐NMMA (Sigma), NorNOHA (Merck‐Millipore), MMP inhibitor (GM6001; Merck‐Millipore), and SB431542 (Cayman Chemicals). Cells were harvested after 3 days of co‐culture and stained with anti‐TCRβ or anti‐CD3‐specific antibodies (Appendix Table S3 ) and analyzed on a LSR Fortessa FACS machine. T cells with CellTrace Violet intensity below freshly stained controls were scored as proliferated T cells.
10
Nor-NOHA Inhibits PDVC57 Tumor Growth
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20–50 μL (depending on tumor size) of 5 mM nor-NOHA (EMD Millipore) in DMSO or DMSO control was applied twice daily, starting one day after injection of PDVC57 cells. Tumor sizes were measured as above every other day. At twenty-one days, the mice were euthanized using carbon dioxide and cervical dislocation. The tumors were then dissected out and weighed.
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