Anti il 1β

Manufactured by BD

Sourced in United States

Anti-IL-1β is a laboratory reagent used to detect and quantify the pro-inflammatory cytokine Interleukin-1 beta (IL-1β) in various biological samples. It functions as a specific antibody that binds to IL-1β, enabling its measurement through immunoassay techniques.

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6 protocols using anti il 1β

Macrophage Responses to N. aromaticivorans

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Macrophages were harvested from the peritoneal cavity of WT and Lgals3^−/− untreated mice using peritoneal lavage. Complete Dulbecco's modified Eagle's medium (6 ml) supplemented with FBS (10%), glutamine (2 mM), 100 U/ml penicillin, and 100 mg/ml streptomycin was injected into the peritoneal cavity and the medium containing cells was retrieved. The extracted cells were depleted of red blood cells using red blood cell lysis buffer containing 8.3 g/l ammonium chloride in 10 mM Tris-HCl, pH 7.5 and washed by centrifugation.
Cells were stimulated with N. aromaticivorans for 24 h (cell/bacteria ratio 1:10) at 37°C in a 5% CO₂ incubator. Where indicated, cells were preincubated with the caspase-1 inhibitor Z-YVAD-FMK (10 μmol/L; Bachem AG, Bubendorf, Switzerland). After incubation, the cell supernatants were collected and cells were labeled with anti-F4/80 (BioLegend), anti-IL-1β (BD Pharmingen), and anti-NLRP3 (R&D systems) fluorochrome-conjugated monoclonal antibodies or isotype matched controls for flow cytometry. The levels of IL-1β and IL-6 in cell supernatants were determined using mouse Duoset ELISA kits (R&D Systems).

Arsenijevic A., Milovanovic J., Stojanovic B., Djordjevic D., Stanojevic I., Jankovic N., Vojvodic D., Arsenijevic N., Lukic M.L, & Milovanovic M. (2019). Gal-3 Deficiency Suppresses Novosphyngobium aromaticivorans Inflammasome Activation and IL-17 Driven Autoimmune Cholangitis in Mice. Frontiers in Immunology, 10, 1309.

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Evaluating Cytokine Production in F3II Cells

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Monolayer F3II cell cultures were treated with trypsin-EDTA (Gibco BRL) for 5 minutes at 37°C.After two washes with MEM 41500 (Gibco BRL) containing 10% FBS (Gibco BRL), the cells were counted using a Neubauer hemocytometer (Merck). A total of 2 × 10⁵ cells in 1 mL of PBS containing 1% bovine serum albumin were added to each well of a 96-well round (U) bottomplate. MHC I and II expression levels were determined after incubating the cells for 20 min on ice with anti-H-2K^dFITC and anti-IA/IE PE antibodies (BD Biosciences, Franklin Lakes, USA). Cytokine production was detected using intracellular staining. Cells were fixed and permeabilized with BD Cytofix/Cytoperm Buffers (BD Biosciences) and incubated with anti-interleukin (IL)-10, anti-IL-1β, anti-transforming growth factor (TGF)-β, anti-IL-6, or anti-tumor necrosis factor (TNF)-α antibodies. All antibodies were purchased from BD Biosciences. For each activation marker, an autofluorescence threshold was established by staining the control cells with the corresponding isotype controls. Staining was detected using a Gallios analyzer flow cytometer (Beckman Coulter, Brea, USA) and analyzed using the Kaluza 1.2 software (Beckman Coulter) for all immunofluorescent flow cytometry investigations.

Fuentes D., Cabezas-Cruz A., Mesa C., Carmenate T., Martínez D., Valdés-Zayas A., Montero E, & Pérez R. (2022). Murine Mammary Carcinoma Induces Chronic Systemic Inflammation and Immunosuppression in BALB/c Mice. Journal of Breast Cancer, 25(3), 218-232.

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Multiparameter Flow Cytometry of Immune Cells

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Single cell suspensions were collected and washed two times with cold PBS and filtered into a 96-well plate for surface staining. Cells were initially stained with pacific orange LIVE/DEAD fixable viability dye (Thermofisher) and then washed two times with cold PBS. Cells were then resuspended in Flow Buffer (PBS, FBS, NaN3, and Hepes Buffer) and Fc-Receptors were blocked with anti-CD16/32 (Biolegend) prior to surface staining. Biotinylated monoclonal antibodies used for surface staining included: Anti-CD3, Anti-CD19, Anti-Ter-119, Anti-NK1.1, Anti-CD11b, Anti-Ly6G, and Anti-Ly6C (Biolegend). Following surface staining, cells were washed twice, and biotinylated antibodies were labeled with streptavidin APC-Cy7. Next, cells were either washed and acquired for surface-only flow cytometry, or were fixed with 2% formaldehyde and then washed/permeabilized with BD perm/wash buffer (BD Biosciences) for intra-cellular flow cytometry. After permeablilization, intra-cellular stains included: anti-IL1β (BD Biosciences), anti-TNF-α (Biolegend), anti-iNOS (affymetrix). After washing, samples were then acquired on a 3-Laser Novocyte Flow Cytometer (Acea Biosciences, Inc.). Data was analyzed using FlowJo software version 10.0 (Treestar, Inc.) and data was compiled using Prism software (GraphPad, Inc.). To verify gating and purity, all populations were routinely back-gated.

Boniakowski A.E., Kimball A.S., Joshi A., Schaller M., Davis F.M., denDekker A., Obi A.T., Moore B.B., Kunkel S.L, & Gallagher K.A. (2018). Macrophage chemokine receptor CCR2 plays a crucial role in macrophage recruitment and regulated inflammation in wound healing. European journal of immunology, 48(9), 1445-1455.

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Multiparametric Flow Cytometry Analysis

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The leukocytes from the spinal cords, spleen, and colonic tissues were collected from the indicated mice. The death cells were excluded first by staining with the LIVE/DEATH Fixable Near-IR Dead Cell Stain Kit (Life Technologies, Eugene, Oregon, USA). For intracellular cytokine staining of IFN-γ, TNF-α, and IL-17 in CD4⁺T cells and IL-1β from F4/80⁺ macrophages, cells were stimulated for up to 10 hours with PMA and ionomycin, and brefeldin A was added at the last 4 hours. Cells were fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences) and incubated with fluorescent antibodies (anti–IL-1β, anti–IFN-γ, anti–TNF-α, and anti–IL-17; BD, Becton Drive, NJ, USA) in the dark at 4°C for 30 min. To measure the expression of phenotype markers on the cell surface, suspended cells were incubated for 1 hour at room temperature in the dark using fluorescent antibodies (anti-CD45, anti-Ly6G, anti-Ly6C, anti-F4/80, and anti-B220; eBioscience, San Diego, CA, USA). A total of 10,000 live cells were analyzed by a FACsAria cytometer (BD, Franklin Lakes, NJ, USA). All the flow cytometry data were analyzed using the CellQuest Pro software, and the gate strategies are shown in fig. S5.

Huang X., Feng Z., Jiang Y., Li J., Xiang Q., Guo S., Yang C., Fei L., Guo G., Zheng L., Wu Y, & Chen Y. (2019). VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1β in macrophages. Science Advances, 5(1), eaau7426.

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Cytokine Profiling in Blood Samples

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Blood samples were drawn at three different moments: at baseline, after 6 months of treatment (T6), and at the end of the follow-up (T12). SPT was carried out at baseline and the end of the follow-up (T12).
For cytokines analysis, peripheral blood was collected and centrifuged at 2,500 rpm for 10 min at 4°C. The serum samples were stored and frozen at −80°C until all analysis. Briefly, wells in a 96-well plate were covered and incubated with purified antibodies anti-IL-17 (R&D System, Minneapolis, MN, EUA), anti-IFN-γ, anti-IL-1β, anti-IL-4, anti-IL-6, anti-IL-8, anti-IL-10, anti-TNF-α, or anti-TGF-β (BD Bioscience, San Diego, CA, EUA), and enzyme-linked immune sorbent assays (ELISA) were carried out, according to the manufacturer's instructions.

de Andrade P.D., Maria e Silva J., Carregaro V., Sacramento L.A., Roberti L.R., Aragon D.C., Carmona F, & Roxo-Junior P. (2022). Efficacy of Probiotics in Children and Adolescents With Atopic Dermatitis: A Randomized, Double-Blind, Placebo-Controlled Study. Frontiers in Nutrition, 8, 833666.

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Cytokine-Induced TSLP Expression in CAFs

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CAFs were seeded at 1.5-3 × 10⁴ cells/well in IMDM 10% FBS in 96-well-plates and starved overnight. The next day, medium was replaced and left for 72 h with the following stimuli: recombinant human IL-1α, IL-1β and TNF-α (R&D System), IL-18 (MBL) (all in IMDM 2% FBS at the indicated concentrations), and the supernatant of necrotic PDAC cells (100 μl). In inhibition experiments, 10 μg/ml anti-IL-1α (R&D System), anti-IL-1β, anti-TNF-α or isotype-matched antibodies (Abs) (BD), and 10 μg/ml Anakinra (Kineret, Amgen Europe) were added. mRNA expression of short and long TSLP isoforms by CAFs untreated or activated by recombinant cytokines was assessed by Real Time PCR (SYBR green, Applied Biosystems) using the primers described in [18 (link)]. In another set of experiments, CAFs were stimulated with the supernatant of THP1 cells treated with the supernatant of tumor cells after treatment with mock-transfected or ASC siRNA or negative control siRNA. TSLP secretion in CAF supernatants was measured by ELISA (DY1398, R&D System).

Brunetto E., De Monte L., Balzano G., Camisa B., Laino V., Riba M., Heltai S., Bianchi M., Bordignon C., Falconi M., Bondanza A., Doglioni C, & Protti M.P. (2019). The IL-1/IL-1 receptor axis and tumor cell released inflammasome adaptor ASC are key regulators of TSLP secretion by cancer associated fibroblasts in pancreatic cancer. Journal for Immunotherapy of Cancer, 7, 45.

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