Whole lung was removed following cardiac perfusion with PBS. Lungs were minced and digested in HBSS containing 1 mg/ml Liberase (0.1 mg/ml final). Cells were filtered using a 70 μm cell strainer and the red blood cells lysed. For IL-33-induced ILC2 activation in vivo, animals were injected intraperitoneally once daily with 500 ng IL-33 (BioLegend) or, as a control, with PBS for four days unless otherwise indicated. In some experiments, mice received daily intraperitoneal injections of IL-33 plus IL-2/anti-IL-2 complexes (IL-2c), made by mixing 1 μg recombinant murine IL-2 (eBioscience) and 5 μg anti-IL-2 monoclonal antibody (JES6-1A12) (Bio X Cell) and incubating for 30 min at 37 °C before co-injection with IL-33 in a final volume of 200 μl in PBS. Lung cells were stained for ILC2 or eosinophils with indicated antibodies, and analyzed using a LSRII or isolated using a FACS Aria III (BD Biosciences). Sort purities were >95%.
Il 33
Manufactured by BioLegend
Sourced in United States
IL-33 is a recombinant human protein that belongs to the IL-1 cytokine family. It is a key regulator of type 2 immune responses and has been implicated in various inflammatory and autoimmune conditions.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 7 (il 7), by BioLegend (11 mentions)
Il 25, by BioLegend (7 mentions)
Interleukin 2 (il 2), by BioLegend (6 mentions)
Il 25, by R&D Systems (5 mentions)
Papain, by Merck Group (4 mentions)
Interleukin 7 (il 7), by R&D Systems (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions)
Rbc lysis buffer, by BioLegend (3 mentions)
Recombinant mouse il 33, by BioLegend (3 mentions)
Easysep mouse pan ilc enrichment kit, by STEMCELL (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Il 13, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Il 12, by BioLegend (2 mentions)
Ifn γ, by R&D Systems (2 mentions)
Il 13, by R&D Systems (2 mentions)
Il 12, by R&D Systems (2 mentions)
78 protocols using il 33
1
ILC2 Activation and Isolation from Murine Lungs
2
Isolation of Lung and Intestinal Cells
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Whole lung was removed following a 5 ml injection of PBS into the right ventricle of the heart. Lungs were minced and digested in HBSS containing 1 mg/ml Liberase with 10 U/ml DNase I (Roche Diagnostics). Cells were filtered using a 70 μm cell strainer and red blood cells lysed. For CD27 labeling, 0.5 mg/ml collagenase type VIII (Sigma) was used. For IL-33 induced ILC2 expansion in vivo, animals were injected intraperitoneally once daily with 500 ng IL-33 (BioLegend) or with PBS for four days.
Colons were removed, opened longitudinally and washed in PBS to remove fecal content. Intestines were cut into two centimeter segments, transferred into HBSS supplemented with 2% FBS and 2 mM EDTA, and shaken for 15 min at 37°C. This step was repeated twice in order to remove epithelial cells and fat tissue. The remaining intestinal tissue was washed, cut in small segments and incubated in digestion media consisting of RPMI 1640, 2% FBS, 0.5 mg/ml collagenase type VIII, 5 U/ml DNase, 100 IU/ml penicillin and 100 μg/ml streptomycin for 40 min at 37°C by gentle shaking. Digested suspensions were filtered, washed in PBS and cells were collected for analysis.
Colons were removed, opened longitudinally and washed in PBS to remove fecal content. Intestines were cut into two centimeter segments, transferred into HBSS supplemented with 2% FBS and 2 mM EDTA, and shaken for 15 min at 37°C. This step was repeated twice in order to remove epithelial cells and fat tissue. The remaining intestinal tissue was washed, cut in small segments and incubated in digestion media consisting of RPMI 1640, 2% FBS, 0.5 mg/ml collagenase type VIII, 5 U/ml DNase, 100 IU/ml penicillin and 100 μg/ml streptomycin for 40 min at 37°C by gentle shaking. Digested suspensions were filtered, washed in PBS and cells were collected for analysis.
3
IL-33-Induced ILC2 Expansion in Mice
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For IL-33-mediated ILC2 expansion, age-matched female mice between 8 and 10 weeks of age were anesthetized with isoflurane and exposed intranasally to 1 μg IL-33 (catalogue number 580506, BioLegend) in 30 μl of PBS on days 0, 1, 2 and 3. Control mice received only PBS. About 16 h later lungs were isolated for analysis as described in the previous section.
4
Bone Marrow-Derived Macrophage Differentiation
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BMDM were differentiated as described previously17 (link). Bone marrow cells from WT mice were cultured in the presence of M-CSF (20 ng ml−1, R&D Systems) in a petri dish (BD OPTILUX). After 7 days, the macrophages (1 × 107 per 10 ml) were stimulated with IL-4 (30 ng ml−1, R&D Systems), IL-13 (30 ng ml−1, R&D Systems), IL-33 (30 ng ml−1, BioLegend) (M2 differentiation); or LPS (1 μg ml−1, E. coli 0111:B4, Sigma-Aldrich), IFN-γ (200 ng ml−1, R&D Systems), M-CSF (5 ng ml−1, R&D Systems) (M1 differentiation); or M-CSF (5 ng ml−1, R&D Systems) alone (M0 macrophages) for 2 days. In some experiments, macrophages were cultured with 10 ng ml−1 IL-4 (R&D Systems) and/or 20 ng ml−1 IL-33 (BioLegend) for 2 days. At the end of cultures, the cells were used for FACS and supernatants for cytokine analysis by ELISA.
5
Murine model of Strongyloides ratti infection
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The S. ratti cycle was maintained in Wistar rats and infections were performed by s.c. infection of 2000 L3 in in 30 μl PBS into the hind footpad of mice as described [49 (link),50 (link)]. Parasite burden in tissue and intestine and quantification of the S. ratti 28S RNA-coding DNA in the faeces of infected mice was performed as described [50 (link)]. For IL-33 treatment 1μg rec IL-33 (Biolegend, Catalogue Nr. 580508) was applied either i.p. in 200 μl PBS or i.n. in 20 μl PBS either 3 h before and 1 day after S. ratti infection or at days 4 and 5 of S. ratti infection. Some mice received 350 μg anti Gr-1 (clone RB6-8C5) at -1 and day 1 of S. ratti infection. Depletion of cells was verified by flow cytometry (S3 Fig ).
6
Isolation of Lung and Intestinal Cells
7
Intranasal Cytokine Treatment for AM Reconstitution
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Recombinant mouse GM-CSF (50 ng, PeproTech), IL-13 (50 ng, PeproTech), CCL2 (6 μg, BioLegend), IL-33 (1 μg, BioLegend), and TGF-β1 (50 ng, PeproTech) were administrated into anesthetized mice via i.n. in a volume of 30 μl of PBS every other day for a total of five times. Recombinant CCL2 was used during the first three times of treatment. The treatment begins from day 18 for AM reconstitution analysis or from day 16 for infection experiment after BMT. Control mice were anesthetized and treated with PBS via i.n.
8
Eosinophil Peroxidase Activity Assay
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Bone marrow-derived eosinophils were incubated with LPS (Sigma-Aldrich), IL-33, and Phorbol 12-myristate 13-acetate (PMA)-ionomycin (Biolegend) for 12 h at the indicated concentration. The EPO activity was measured by the spectrophotometric method (31 (link)). Briefly, 100 µl of culture supernatant from each sample was placed in a 96-well plate, and 100 µl of substrate solution containing 0.1 µM o-phenylenediamine-dihydrochloride, 0.1% Triton X-100, and 1 µM hydrogen peroxide (Sigma-Aldrich) was added in each well. After incubation for 30 min at 37°C, the enzymatic reaction was stopped by adding 50 µl of 4 M sulfuric acid. Absorbance was measured at 492 nm using a Multiscan JX system (Thermo Fisher Scientific).
9
Induction of Airway Inflammation in Mice
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For IL-33-induced airway inflammation, mice were anesthetized using isoflurane and treated three times intranasally or intratracheally every other day with 0.5 µg IL-33 (BioLegend, USA) or PBS (GIBCO Life Technologies, USA) as described previously (17 (link)). Mice were sacrificed at the indicated time points after the final IL-33 administration.
For acute HDM-induced allergic airway inflammation, mice were anesthetized using isoflurane and sensitized by intranasal or intratracheal injection with 1 or 10 µg HDM extract (Greer, SC, USA) or PBS. After 7 days, animals were challenged daily on days 0–4 with 10 µg HDM intranasally or intratracheally and sacrificed at the indicated time points after the final treatment as described previously (46 (link)). For chronic HDM-induced allergic airway inflammation, mice were anesthetized using isoflurane and treated intranasally three times weekly for 5 weeks with 25 µg HDM or PBS and sacrificed at the indicated time points after the final treatment [adapted from Ref. (47 (link))].
For acute HDM-induced allergic airway inflammation, mice were anesthetized using isoflurane and sensitized by intranasal or intratracheal injection with 1 or 10 µg HDM extract (Greer, SC, USA) or PBS. After 7 days, animals were challenged daily on days 0–4 with 10 µg HDM intranasally or intratracheally and sacrificed at the indicated time points after the final treatment as described previously (46 (link)). For chronic HDM-induced allergic airway inflammation, mice were anesthetized using isoflurane and treated intranasally three times weekly for 5 weeks with 25 µg HDM or PBS and sacrificed at the indicated time points after the final treatment [adapted from Ref. (47 (link))].
10
Intranasal IL-33 and Retinoic Acid
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Anesthetized mice were treated intranasally with 20ul of IL-33 (250 ng) (Biolegend, San Diego, CA, USA) on days 0, 1, and 2, and the mice were analyzed on day 3. For administration of retinoic acid, a total of 250 μg of all-trans-RA (Sigma-Aldrich, St. Louis, MO, USA) in 30 µl of DMSO was administered intraperitoneally to mice every day for 6 days. Three days after RA administration, RA received mice were simultaneously treated intranasally with 250 ng of IL-33 on days 4, 5, and 6, and the mice were analyzed on day 7.
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