L nmma

Manufactured by Cayman Chemical

Sourced in United States

L-NMMA is a chemical compound used as a tool in biochemical research. It functions as a nitric oxide synthase inhibitor, which helps in the study of the role of nitric oxide in biological systems.

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Lab products found in correlation

Nor noha, by Cayman Chemical (5 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (3 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (3 mentions) L nac, by Merck Group (1 mentions) Gm csf, by Thermo Fisher Scientific (1 mentions) L arginine, by Merck Group (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Hydrogen peroxide h2o2, by Merck Group (1 mentions) Anti cd3 28 dynabeads, by Thermo Fisher Scientific (1 mentions) Fluorescence activated cell sorting (facs), by Merck Group (1 mentions) Anti ifn γ clone xmg1, by BioXCell (1 mentions) 1 methyl dl tryptophan 1 mt, by Merck Group (1 mentions) Cd3 clone 145 2c11, by Thermo Fisher Scientific (1 mentions) Cd28 clone 37, by Thermo Fisher Scientific (1 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Cayman Chemical (1 mentions) Syringe pump, by Harvard Apparatus (1 mentions) Celecoxib, by Abcam (1 mentions) 1 methyl dl tryptophan, by Merck Group (1 mentions) Dynabeads human t cell activator cd3cd28, by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)

12 protocols using l nmma

Tumor-Induced MDSC Generation and Modulation

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BM-MDSCs were generated by culturing BM cells for 4 d in the presence of GM-CSF (40 ng/ml; Peprotech). BM-MDSCs were exposed to tumor-derived factors by the addition of 40% B16F10 TES, IL-6 (40 ng/ml; Peprotech), hydrogen peroxide (H₂O₂; 250 mM; Sigma-Aldrich), and l-arginine (1 mM; Sigma-Aldrich) during the last 24 h of culture. In another assay, BM-MDSCs were cultured with or without 40% TES, 1 mM L-NAC (Sigma-Aldrich), 100 µM L-NMMA (Cayman Chemical), or 100 µM nor-NOHA (Cayman Chemical). TES was produced by culturing B16F10 tumor cells, freshly prepared from B16F10 tumors, at 1 × 10⁷ tumor cells/ml for 24 h, and stored at −80°C until use.

Yan D., Wang J., Sun H., Zamani A., Zhang H., Chen W., Tang A., Ruan Q., Yang X., Chen Y.H, & Wan X. (2019). TIPE2 specifies the functional polarization of myeloid-derived suppressor cells during tumorigenesis. The Journal of Experimental Medicine, 217(2), e20182005.

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Suppressor Cell-Mediated T Cell Inhibition

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Splenic CD8⁺ T cells were purified as described above, labelled with 0.5mM CFSE (Life Technologies) and plated at 1×10⁵ per well as responder cells for in vitro stimulation. T cells were activated by either anti-CD3/28 dynabeads per manufacturer's instructions (Invitrogen), or by 5×10⁵ irradiated donor (BALB/c) APCs. FACS sorted suppressor cells were added at a 1:1 ratio with the CFSE-labeled T responder cells, and incubated at 37°C for 96hours. T cell proliferation was measured by CFSE dilution. For indicated studies, FACS sorted suppressor cells were either pretreated at room temperature for 30 minutes with 10μg/ml anti-IFN-γ (clone XMG1.2, BioXCell) prior to addition to the proliferation assays, or added to the proliferation assays in the presence of 5mM L-NMMA or D-NMMA (Cayman Chemical,) or 2mM 1-Methyl-DL-tryptophan (1-MT) (Sigma-Aldrich) or vehicle (2% carboxymethylcellulose) For suppression assays by graft Gr1^HI and Ly6C^HI cells, CFSE labeled responder CD8⁺ T cells were plated at 1×10⁴ per well, co-cultured with 1×10⁴ anti-CD3/28 dynabeads or 5×10⁴ BALB/c APCs, and 1×10⁴ FACS sorted suppressor cells from the graft. T cell proliferation was determined by CFSE dilution after 96 hours.

Bryant J., Lerret N.M., Wang J.J., Kang H.K., Tasch J., Zhang Z, & Luo X. (2014). Preemptive donor apoptotic cell infusions induce IFN-γ-producing myeloid derived suppressor cells for cardiac allograft protection. Journal of immunology (Baltimore, Md. : 1950), 192(12), 6092-6101.

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Inhibition of NO Synthesis Impacts C2C12 Parasite Internalization

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C2C12 cells (1 × 10⁴) were infected as described in In Vitro Multiplication Assay, and then 6 µM of L-NMMA (Cayman, USA), an NO synthesis inhibitor, was added to the wells. After 72 hpi, the cells were fixed with 4% PFA for 15 min and incubated with DAPI as described previously. The number of internalized parasites in 100 cells/coverslip was counted.

Silva N.S., Orikaza C.M., de Santana F.R., dos Santos L.A., Salu B.R., Oliva M.L., Sinigaglia R.D, & Mortara R.A. (2021). Interleukin-9 in Immunopathology of Trypanosoma cruzi Experimental Infection. Frontiers in Cellular and Infection Microbiology, 11, 756521.

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Evaluating T-cell Proliferation Dynamics

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T-cells isolated from naïve B6 spleens were stained with 5 μM carboxyfluorescein succinimidyl ester (CFSE; Sigma) and stimulated using plate-bound CD3 (clone 145-2C11, eBioscience) and CD28 (clone 37.51, eBioscience) antibodies(26 ). For antigen-specific experiments, sorted OT-1 splenic T-lymphocytes were exposed to irradiated (20 Gy) naïve splenocytes pulsed with OVA_257-264 (SIINFEKL; 1 μg/ml; InVivoGen). Where indicated, T-cells were co-cultured with MDSCs, IPI-145, nor-NOHA, and/or L-NMMA (Cayman Chemicals, 300 μM each) for 4 hours prior to stimulation, and flow cytometry was used to quantify 72-hour CFSE dilution. Proliferation was quantified as the average number of divisions for all cells in the culture (division index) using FlowJo software(27 (link)). Media for all functional immune assays consisted of RPMI 1640 supplemented with 10% FCS, 2 μM β-ME, HEPES, nonessential amino acids, glutamine, and antibiotics.

Davis R.J., Moore E.C., Clavijo P.E., Friedman J., Cash H., Chen Z., Silvin C., Van Waes C, & Allen C. (2017). Anti-PD-L1 efficacy can be enhanced by inhibition of myeloid derived suppressor cells with a selective inhibitor of PI3Kδ/γ. Cancer research, 77(10), 2607-2619.

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Microfluidic Platform for HUVEC Culture

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Translucent tubes with 0.8 mm inner diameter (Cole-Parmer) connected with barbed elbow and T-connectors (Cole-Parmer) were used to perfuse the microfluidic platform. The assembled tubing circuits were autoclaved using hot steam sterilization and connected to the inlet and outlet ports on the microfluidic devices seeded with HUVECs. The inlet port was connected to an EGM reservoir and the outlet port was connected to 10 mL BD-syringes (Fisher Scientific) mounted on a syringe pump (Harvard Apparatus). The intravascular pressure in the perfusion microchannels was adjusted via the hydrostatic pressure head of the media reservoir. To examine the role of nitric oxide, L-MMMA (Cayman Chemical) was reconstituted in DMSO for long term storage and diluted in EGM to make perfusion media supplied with 200 μM L-NMMA.

Akbari E., Spychalski G.B., Rangharajan K.K., Prakash S, & Song J.W. (2018). Flow dynamics control endothelial permeability in a microfluidic vessel bifurcation model. Lab on a chip, 18(7), 1084-1093.

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Modulating CD8+ T cell activation

Cited 1 time

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Naïve CD8⁺ T cells (1 × 10⁵ cells/well) labeled with CFSE (Invitrogen; labeled according to the manufacturer’s protocol) were stimulated with CD3/CD28 Human T cell-Activator Dynabeads (5 μl/ml; Invitrogen) in the presence or absence of MDSCs (0.25 × 10⁵ cells/well) and/or IL18/IFNα-activated NK cells (0.25 × 10⁵ cells/well) in 96-well plates. As an alternative method for CD8⁺ T cell expansion, CFSE-labeled naïve CD8⁺ T cells (1 × 10⁵ cells/well) were stimulated with staphylococcal enterotoxin B-pulsed mature monocyte-derived DCs (1 × 10⁴ cells/well; matured for 48 h with 50 ng/ml TNFα), as previously described (18 (link)). When indicated, cells were co-cultured in the additional presence of small-molecule inhibitors or blocking antibodies against suppressive factors. On day 4–6, expanded CD8⁺ T cells were analyzed for proliferation via CFSE dilution and intracellular granzyme B expression. The following inhibitors/blocking antibodies were used in this study: celecoxib (20 μM; BioVision), 1-methyl-DL-tryptophan (1 mM; Sigma-Aldrich), L-NMMA (200 μM; Cayman Chemical), IL10 mAb (clone 25209; 1 μg/ml; R&D Systems), and nor-NOHA (200 μM; Cayman Chemical). The concentrations used did not affect viability in cell cultures, as confirmed by live cell counts.

Wong J.L., Obermajer N., Odunsi K., Edwards R.P, & Kalinski P. (2016). Synergistic COX2 induction by IFNγ and TNFα self-limits type-1 immunity in the human tumor microenvironment. Cancer immunology research, 4(4), 303-311.

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Intracellular Bacterial Quantification in Macrophages

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Bone marrow derived macrophages were infected with the indicated multiplicity of infection (MOI) of Fn or SchuS4 as previously described (Griffin et al., 2013 (link)). Briefly, bacteria were diluted to the indicated MOI and added to BMM. BMM were incubated for 90 min at 37^∘C/5% CO₂. Then, bacteria containing medium was pipetted off and BMM were incubated with gentamicin (50 μg/ml) for 45 min. BMM were washed extensively with PBS and incubated in DMEM supplemented with 10% heat inactivated fetal bovine serum, L-glutamine, non-essential amino acids, and HEPES (cDMEM; all from Life Technologies). Intracellular bacteria were enumerated by lysing BMM with water and plating lysates on MMH agar as previously described (Bauler et al., 2011 (link)). Where indicated cells were pretreated with 3 mM N-aceytlcysteine (NAC; Sigma), 1 mM L-NMMA (Cayman Chemicals, Ann Arbor, MI, USA) or 250 μM mitoTempo (Enzo Life Sciences, Farmingdale, NY, USA) 16, 24, or 1 h prior to infection, respectively. Effective concentrations of NAC and L-NMMA used in this study were determined by their ability to inhibit production of ROS and RNS in BMM treated with LPS + IFN-γ (Crane, unpublished data). Various concentrations of mitoTempo were tested in BMM. Only concentrations of 250 μM and above had any effect on ROS production in cells (Bauler, unpublished data).

Crane D.D., Bauler T.J., Wehrly T.D, & Bosio C.M. (2014). Mitochondrial ROS potentiates indirect activation of the AIM2 inflammasome. Frontiers in Microbiology, 5, 438.

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Calcium Signaling Modulators Protocol

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KN93 (Santa Cruz Biotechnology), H89 (Sigma-Aldrich), l-NMMA (Cayman Chemical Company, Ann Arbor, MI, USA), EGTA-AM (Life Technologies), BAPTA-AM (Sigma-Aldrich) and BAPTA (Sigma-Aldrich) were purchased. These chemicals were dissolved in dimethyl sulfoxide or double-distilled water.

Tsoi H., Lam K.C., Dong Y., Zhang X., Lee C.K., Zhang J., Ng S.C., Ng S.S., Zheng S., Chen Y., Fang J, & Yu J. (2017). Pre-45s rRNA promotes colon cancer and is associated with poor survival of CRC patients. Oncogene, 36(44), 6109-6118.

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Adoptive T-cell Transfer in LLC-OVA Tumor Model

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LLC‐OVA cells (2 × 10⁶ per mouse) were injected subcutaneously into the flanks of 6–8 week‐old female NOD‐SCID mice (Charles River) on day 0. At the same time, OT‐I cells were cultured alone or cocultured with TAMos derived from spleens of LLC tumor‐bearing mice in the presence or absence of L‐NMMA (500 µg mL⁻¹) (Cayman) under stimulation of OVA_257‐264 peptides for 48 h. The activated T cells were then expanded with IL‐2 (10 U mL⁻¹) for a further four days. Then, T cells (3 × 10⁶ per mouse) were intravenously transferred into LLC‐OVA tumor‐bearing mice on day 7 after tumor inoculation. On the indicated day after T cell transfer, tumor‐bearing mice were euthanized and tumors, spleens, and TDLNs were excised, followed by flow cytometry analysis.^[⁵³
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Yang Z., Liu L., Zhu Z., Hu Z., Liu B., Gong J., Jin Y., Luo J., Deng Y., Jin Y., Wang G, & Yin Y. (2024). Tumor‐Associated Monocytes Reprogram CD8+ T Cells into Central Memory‐Like Cells with Potent Antitumor Effects. Advanced Science, 11(16), 2304501.

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Inhibition of T-cell Proliferation by G-MDSCs

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T-lymphocytes were isolated from spleens of naive Balb/c mice. 1 × 10⁶ cells/mL T-lymphocytes were stimulated by CD3/28 antibodies and then stained with 5 mM carboxyfluorescein succinimidyl ester (CFSE, Sigma). For antigen-specific experiments, T-lymphocytes were exposed to irradiated naive splenocytes (20 Gy) pulsed with OVA_257-264 (SIINFEKL, 1 µg/mL; InVivoGen). T-lymphocytes were co-incubated with sorted G-MDSCs, SNA (1 µM), nor-NOHA or L-NMMA (300 µM each, Cayman Chemicals) for 4 h prior to stimulation, and quantified by flow cytometry 72 h later. Proliferation was quantified as the average number of divisions for all cells. The formula: (divisions of non-treated group - divisions of treated group)/divisions of non-treated group × 100% was used to calculate the inhibition rate of T-lymphocyte proliferation.

Shi X., Li X., Wang H., Yu Z., Zhu Y, & Gao Y. (2018). Specific inhibition of PI3Kδ/γ enhances the efficacy of anti-PD1 against osteosarcoma cancer. Journal of Bone Oncology, 16, 100206.

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