A liver sample (50 mg) from the NCD- and MCD-fed mice was subjected to identification and quantification of the 65 FFAs using a Q300 Metabolite Array Kit (Metabo-Profile, Shanghai, China). CD3+ T cells were enriched from C57BL/6 mouse spleens by a mouse CD3+ T-cell enrichment column (R&D Systems, Minneapolis, MN). Isolated CD3+ T cells were cultured in wells precoated with the anti-CD3 monoclonal antibody (5 μg/mL; BD Biosciences) and the anti-CD28 monoclonal antibody (2 μg/mL; BD Biosciences) and then with 25 or 50 μmol/L ADA, AA (MCE, Monmouth Junction, NJ), or dimethyl sulfoxide at 37°C with 5% CO2 for 48 hours, and 25 μmol/L ADA group was sent to transcriptome sequencing after extracting RNA by RNasey Plus Micro Kit according to instructions (Qiagen, Hilden. Germany). Then, flow cytometry was conducted to assess apoptosis and cell surface markers of DNT cells. One μmol/L, 5 μmol/L AKT activator SC-79 (MCE) or 1 nmol/L, 5 nmol/L NF-κB signaling pathway inhibitor BAY 11-7082 (MCE) were used to interrupt effect induced by AA in TCRαβ+ DNT cells or TCRγδ+ DNT cells for 48 hours.
Anti cd3 monoclonal antibody
Manufactured by BD
Sourced in United States
The Anti-CD3 monoclonal antibody is a laboratory reagent used in immunological research. It binds to the CD3 complex on the surface of T cells, which is essential for T cell activation and function. This product can be used in various in vitro and ex vivo applications to study T cell biology and immune response.
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Lab products found in correlation
Anti cd28 monoclonal antibody, by BD (2 mentions)
Zm241385, by MedChemExpress (2 mentions)
Anti cd28, by BD (2 mentions)
Interleukin 2 (il 2), by BD (2 mentions)
Mouse cd3 t cell enrichment column, by R&D Systems (1 mentions)
Rnasey plus micro kit, by Qiagen (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
96 well round u bottom plate, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ mab, by BD (1 mentions)
Intrapre reagent, by Beckman Coulter (1 mentions)
Leukocyte activation cocktail, by BD (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Beijing Chemical Works (1 mentions)
Phosphate buffered saline (pbs), by Boster Bio (1 mentions)
Adenosine, by Merck Group (1 mentions)
11 protocols using anti cd3 monoclonal antibody
1
Identifying Fatty Acids and Immunomodulation in Liver Disease
2
Cytokine Production in Pseudomonas-Infected Mice
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Spleens and mediastinal lymph nodes (MLNs) from P. aeruginosa infected mice were harvested and placed in complete Dulbecco’s modified Eagle’s medium (cDMEM). Lymphocyte suspensions were prepared from the MLNs and spleens by pressing the cells through a 70-μM nylon cell strainer (Falcon; BD Labware). Red blood cells were lysed. The cell number was counted using the Countess Automated Cell Counter. Cells (5 × 106 cells/ml) were cultured in a 48-well plate pre-coated with anti-CD3 monoclonal antibody ((BD Pharmingen, 5 μg/ml), and culture supernatants were collected after 72 h of stimulation. Culture supernatants were analyzed for the levels of cytokines present via ELISA as previously described11 (link).
3
Measuring Treg Suppressive Activity on Tconvs
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Similar to a previous study, we used the proliferation of carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA)-labeled CD4+CD25- T-cells (Tconvs) to measure the regulatory activity of Tregs (18 (link)). Briefly, cells were rinsed with PBS and treated with 1 μM of CFSE for 8 min to be labeled with CFSE at 37°C. Then, to remove any remaining CFSE, the cells were washed twice with fetal bovine serum. Tregs were isolated as described above from sepsis mice. Inhibition of effector T-cell proliferation was used to assess regulatory activity of Tregs. Tconvs were isolated from the spleens of C57BL/6 mice with the FACSAria IIu instrument and labeled with CFSE. Tconvs (1×105 cells) were co-cultured with Tregs in a 1:1 ratio. Tconv cell proliferation was induced with anti-CD3 monoclonal antibody (1 μg/mL; BD Biosciences), anti-CD28 (1 μg/mL; BD Biosciences), and IL-2 (5 μg/mL; BD Biosciences) for 4 days in a 48-well plate, and after CD4+CD25- gating, the degree of Tconv proliferation was examined (25 (link)). In another set of experiments, ZM241385 (1 μM; MedChem Express) was added.
4
Macrophage-CD4+ T Cell Coculture
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Macrophages were suspended in complete media containing 100 ng/mL LPS, seeded at 20,000 cells/well in a 96-Well Round (U) Bottom Plate (Thermo-Fischer), and incubated overnight at 37ºC, 5% CO2. CD4+ T cells obtained from the same donor as the monocytes were thawed, stained with 5, 6-carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Carlsbad, CA), and co-incubated with the macrophages at a ratio of 1:1 in the presence of 1 μg/mL anti-CD3 monoclonal antibody (BD Pharmingen, San Diego, CA) for 3 days. Supernatants were collected and the cells were analyzed by flow cytometry.
5
Quantifying IFN-γ Production in NK and T Cells
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Leukocytes were stained with surface markers, fixed, permeabilized with IntraPre Reagent (Beckman Coulter, Fullerton, CA, USA), and further stained with the following antibodies against intracellular markers: anti-CD3 monoclonal antibody (mAb), anti-CD56 mAb, and anti-IFN-γ mAb (BD Biosciences). The FlowJo software program (version 10, FLOWJO, BD Biosciences) was used for data analysis. For the measurement of intracellular cytokine production, cells were stimulated at 37 °C for 5 h with Leukocyte Activation Cocktail (BD Biosciences) before staining, as previously described [29 (link)]. When we checked cell viability after thawing of frozen cells, the viability were good with more than 75%. Cell viabilities were approximately checked 65% using dead cell marker. (Additional file 2 : Fig. 2). We analyzed IFN-γ positive NK cell population and the gating process was summarized at Fig. 1 A. Finally, T-cell producing IFN-γ was analyzed (Additional file 3 : Fig. 3).![]()
The gating process of flow cytometric analysis (
6
Evaluating T-cell Proliferation with hMSCs
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Isolated CD3+ T lymphocytes were stained using a CellTrace CFSE cell proliferation kit (5 mmol/L; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Labeled CD3+ T cells were cultured in replicate wells with or without hMSCs and stimulated with an anti-CD3 monoclonal antibody (0.2 mg/mL, BD Biosciences, San Diego, CA) and an anti-CD28 monoclonal antibody (1 mg/mL, BD Biosciences, San Diego, CA) for 96 h. T-cell proliferation was evaluated by flow cytometric analysis of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilutions.
7
Amplification of Peptide-Specific CTLs
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Peptide-specific CTLs obtained from ELISPOT-positive wells after in vitro CTL induction were expanded as described previously [42 (link), 43 (link)]. Suspensions of the remaining peptide-specific CTLs were co-cultured with 5 × 106 MMC-treated (30 μg/ml at 37° C for 30 min) Jiyoye and EB-3 cells in 25 ml of 5% Auto serum/AIM-V containing 40 ng/ml anti-CD3 monoclonal antibody (BD Pharmingen, San Diego, CA) on day 0. IL-2 was added (final concentration: 120 IU/ml) on day 1, and 5% Auto serum/AIM-V containing 30 IU/ml IL-2 was provided on days 5, 8, and 11. On day 14, expanded CTL activity was examined using an IFN-γ ELISPOT assay.
8
Isolation and Activation of Mouse CD4+ T Cells
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To isolate CD4+ T cells from mouse spleens, positive magnetic sorting was performed using an AutoMACS separator (130-104-454, MACS CD4+ microbeads; Miltenyi Biotech, Bergisch Gladbach, Germany). Fluorescence-labeled anti-CD4, anti-CD8, and anti-CD19 monoclonal antibodies (BD Biosciences) were used to verify isolated CD4+ population purity using flow cytometry. After purification, 2×106 cells/mL CD4+ T cells were resuspended in RPMI 1640 supplemented with 1% v:v NEAA, 10% v:v FBS, 2 mM L-glutamine, 1% v:v sodium pyruvate, 10 mM HEPES, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol, and 100 U/mL penicillin, then seeded at 2×105 cells/well in flat-bottomed 96-well microtiter plates coated with 5 μg/mL anti-CD3 monoclonal antibody (555273; BD Biosciences). Cells were cultured for 72 hours, before collection of culture super-natants for cytokine evaluations using BD cytometric bead arrays (552364 mouse inflammation kit; BD Biosciences).
9
Expansion of Peptide-Specific CTLs for ELISA
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The peptide-specific CTLs harvested from ELISPOT-positive wells after in vitro CTL induction were expanded by a modified protocol based on the previously described methods [30] , [31] (link). A total of 5×104 CTLs was cultured with 5×106 MMC-inactivated Jiyoye or EB-3 cells (30 µg/ml at 37°C for 30 min treatment) in 25 ml of AIM-V/5% AS containing 40 ng/ml of anti-CD3 monoclonal antibody (BD Biosciences, San Diego, CA) on day 0. IL-2 was added 24 h later (final concentration: 120 IU/ml), and fresh AIM-V/5% AS containing 30 IU/ml of IL-2 was provided on days 5, 8 and 11. On day 14, CTLs were harvested and the CTL activity was examined by an IFN-γ enzyme-linked immunosorbent assay (ELISA).
10
Immunofluorescence Analysis of AAA Tissue
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126 patients from CMUaB were examined by an experienced vascular surgeon within 2 days before and after the OSR. Until December 2018, nine patients not willingly and openly reveal their inpatients of information via publication, whether their clinical data, aneurymal tissue, or blood sample. Finally, aneurysm specimens from 96 male patients were eligible for further analyses. Here, AAA tissue specimen (n=17) and corresponding peripheral blood samples (n=17) were obtained who underwent OSR. All samples were harvested at the most dilated place of aortaventralis, by which tissues in vitro were washed in PBS (Boster, Wuhan, China), opened transversely, and fixed in 4% paraformaldehyde (Beijing Chemical Works, Beijing, China) for 24 hrs. The samples were then embedded in paraffin and sectioned transversely at 2–3 µm continuously to make parallel sections. Immunofluorescence was stained by anti-CD3 monoclonal antibody (BD Biosciences, San José, CA, USA) to identify T lymphocytes on one section for cell type analysis and localization and the other was prepared for fluorescence in situ hybridization (FISH).
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