For the administration of cytokines, a volume of 5 μl containing 10 ng of recombinant murine IL-1β (R&D Systems) [15 (link)] in PBS or 5 μl of collagenase type IV (Worthington Biochemical Corp., Lakewood, NJ, USA) was administered by IA injection into the right knees as previously described [20 (link)]. Briefly, the region of the knee joint was shaved and disinfected. The joint was flexed at 90 degrees to provide access to the joint cavity at the lower pole of the patella. The injection was performed by puncturing the knee joint laterally from the patellar ligament using a 32-gauge microneedle (Hamilton AG, Bonaduz, Switzerland). The left knee joint was treated with the same volume of PBS to serve as a control. After the procedure, the animals were left for 48 h prior to IV injection of SFs to incubate the cartilage with the corresponding cytokine/PBS. For all IA injections, a stereomicroscope was used (Stemi 2000-C; Carl Zeiss Microscopy, Oberkochen, Germany).
Recombinant murine il 1β
Manufactured by R&D Systems
Sourced in United States
Recombinant murine IL-1β is a cytokine that is produced in Escherichia coli. It is a member of the interleukin 1 cytokine family.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase type 4, by Worthington (1 mentions)
Stemi 2000 c, by Zeiss (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Thermo Fisher Scientific (1 mentions)
Il 12p70, by Thermo Fisher Scientific (1 mentions)
Il 23, by R&D Systems (1 mentions)
Recombinant murine ifn γ, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Proteintech (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Lipopolysaccharides (lps), by Biosharp (1 mentions)
P foxo1, by Cell Signaling Technology (1 mentions)
Gapdh, by Boster Bio (1 mentions)
F4 80, by Proteintech (1 mentions)
Col2a1, by ABclonal (1 mentions)
Iκb kinase ikk β, by Cell Signaling Technology (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Cd206, by Abcam (1 mentions)
11 protocols using recombinant murine il 1β
1
Intra-articular Cytokine Administration
2
Quantifying Murine IL-1 Cytokines
3
Cytokine Modulation by Treponema Outer Vesicles
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In 96-well cell culture plates, 1 ng/ml of recombinant murine IL-1β (R&D Systems), IL-6 (Peprotech), IL-23 (R&D Systems), and IL-12p70 (Peprotech) were incubated with OMVs (1 and 10 μg/ml) in 200 μl/well RPMI 1640 complete medium for 24 h at 37°C in humidified aerobic conditions (5% CO2). BMDCs were stimulated with 100 ng/ml Pam3CSK4 for 24 h at 37°C. Culture supernatants of Pam3CSK4-treated BMDCs were incubated with T. denticola OMVs in the presence or absence of 2 mM phenylmethylsulfonyl fluoride (PMSF, Thermo Fisher Scientific), a serine protease inhibitor, for 1 h. In addition, T. denticola OMVs were heated at 95°C for 10 min and incubated with culture supernatants of Pam3CSK4-treated BMDCs for 1 h. The levels of the remaining cytokines in the medium or culture supernatants were measured by ELISA.
4
Signaling Pathways in Macrophage Activation
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Recombinant murine IL-1β and recombinant murine M-CSF protein were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant murine IFN-γ was purchased from Peprotech (Rocky Hill, NJ, USA). Lipopolysaccharides (LPS) were obtained from Biosharp (Beijing, China). Metformin hydrochloride was purchased from CTAO (Eugene, OSU, USA). Anti-p85-PI3K, MTOR, p-MTOR, FoxO1, p-FoxO1, AKT, p-AKT, GSK3ß, p-GSK3ß, NF-κB, p-NF-κB, IκBα, p-IκBα, IκB kinase (IKK)-β, and p-IKKα/β antibodies were supplied by Cell Signaling Technology (Beverly, MA, USA). MMP3, MMP13, INOS, and CD206 antibodies were obtained from Abcam (Cambridge, UK). Col2a1 and Sox9 antibodies were purchased from Abclonal (Wuhan, China). IL-1ß, IL-6, CD86, and F4/80 antibodies were purchased from Proteintech (Wuhan, China). GAPDH, ß-Actin, and secondary antibodies were purchased from Boster (Wuhan, China).
5
IL-1β Treatment of Cells
6
Quantification of Murine IL-1β by ELISA
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A monoclonal antibody to IL-1β (R&D systems) was diluted to 2 μg/ml in carbonate buffer and dispensed in 96-well Maxisorb Immunoplates (Nunc, Roskilde, Denmark) overnight. Samples were added for one hour, followed by the secondary antibody (a biotinylated goat anti-mouse IL-1β (R&D systems)). Extravidin-alkaline phosphatase (1:1,000, Sigma) was added for 1 hour after which 1 mg/ml 4-nitrophenyl phosphate substrate (Sigma) was added. After 20 minutes, plates were read at 450 nM and results were calculated by reference to a standard curve of recombinant murine IL-1β (R&D systems).
7
Osteoclastogenesis Regulation Protocol
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Sudachitin was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). LPS derived from Escherichia coli (O55:B5) was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human M-CSF was kindly provided by the Morinaga Milk Industry Co. (Tokyo, Japan). Recombinant murine soluble RANKL (sRANKL) and recombinant murine IL-1β were obtained from R&D Systems (Minneapolis, MN) and PeproTech (Rocky Hill, NJ), respectively. Prostaglandin E2 (PGE2) was obtained from Sigma-Aldrich. The anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-SAPK/JNK, anti-SAPK/JNK, anti-phospho-IκB, and anti-IκB antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-c-fos and anti-NFATc1 antibodies were obtained from Santa Cruz Biotechnology (San Diego, CA). The anti-cathepsin K, anti-DC-STAMP (clone 1A2) and anti-Atp6v0d2 antibodies were purchased from BioVision (Mountain View, CA), MILLIPORE (Temecula, CA) and AVIVA Systems Biology (San Diego, CA), respectively.
8
Murine IL-1β Signaling in Neurons and Astrocytes
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Cells were treated with recombinant murine IL-1β (R&D Systems, Minneapolis, MN, USA) for either 24 or 48 h in an incubation buffer of MS for mixed cultures and NB for neurons both supplemented with the vehicle, 0.1% fatty-acid-free bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA). As both neurons and astrocytes have functional IL-1R1 receptors, higher concentrations for the mixed cultures were utilized to ensure optimal signaling (Birgit Fogal and Sandra J Hewett, unpublished observations).
9
Modulation of Plasma Cell Homeostasis
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To assess the effect of FTY720 treatment on plasma cell accumulation and expansion, mice were intra-peritoneally (i.p.) administered with PBS or 100ug FTY720 (Millipore Sigma). At start of the experiment, mice were orally treated with mifepristone (RU486) as described above, with i.p. treatment of PBS or FTY720. Subsequently, PBS or FTY720 dosing was continued every second day till end of the experiment on Day 9.
Salmonella typhimurium wildtype strain SB300A1[18 , 49 (link)] were grown with shaking overnight at 37 °C in Luria-Bertani (LB) broth, sub-cultured for 4 h, and washed twice with cold PBS prior to use. For oral Salmonella infections, mice were deprived of food for 4 h and gavaged orally with 5 X 107 CFU of bacteria in 200μl PBS. Mice were kept without food and water for 1 h after infection. The final infection dose was verified by plating serial dilutions of the bacterial suspension on LB agar plates.
To inhibit GAPs using IL-1β treatment, mice were treated i.p. with 100 ng of recombinant murine IL-1β (R&D Systems, Minneapolis, MN). At start of the experiment, mice were orally treated with mifepristone (RU486) as described above for 4 consecutive days. On day zero, MyD88f/f and MyD88f/f Math1Cre*PR mice were treated with IL-1β or PBS, concomitantly with RU486.
Salmonella typhimurium wildtype strain SB300A1[18 , 49 (link)] were grown with shaking overnight at 37 °C in Luria-Bertani (LB) broth, sub-cultured for 4 h, and washed twice with cold PBS prior to use. For oral Salmonella infections, mice were deprived of food for 4 h and gavaged orally with 5 X 107 CFU of bacteria in 200μl PBS. Mice were kept without food and water for 1 h after infection. The final infection dose was verified by plating serial dilutions of the bacterial suspension on LB agar plates.
To inhibit GAPs using IL-1β treatment, mice were treated i.p. with 100 ng of recombinant murine IL-1β (R&D Systems, Minneapolis, MN). At start of the experiment, mice were orally treated with mifepristone (RU486) as described above for 4 consecutive days. On day zero, MyD88f/f and MyD88f/f Math1Cre*PR mice were treated with IL-1β or PBS, concomitantly with RU486.
10
Murine IL-1β Signaling Pathway Modulation
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Recombinant murine IL-1β was bought from R&D Systems (Minneapolis, MN, USA). PD98059, SB203580, and SP600125 were obtained from Biomol (Plymouth Meeting, PA, USA). Bradford reagent was from Bio-Rad (Richmond, CA, USA). Dexamethasone and RU486 were obtained from Sigma (St. Louis, MO, USA). DMEM and gentamicin were purchased from Gibco-BRL (Gaithersburg, MD, USA).
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