For N‐formyl peptide culture, the cerebral vessels from young (2–3 months old) WT mice were dissected, harvested, and washed in PBS. Cerebral vessels from left and right hemisphere were kept separate from each mouse brain and were randomized for 30‐minute culture in PBS with either 10 μmol/L N‐formyl peptide (N‐Formyl‐Nle‐Leu‐Phe‐Nle‐Tyr‐Lys; Genscript catalog No. RP12959) or vehicle (PBS+1% dimethyl sulfoxide), similar to what was previously done.15 After 30‐minute culture, vessels were washed twice in PBS and immediately flash frozen for immunoblot analysis. For recombinant IL‐6 culture, the cerebral vessels from young (2–3 months old) mice were dissected, harvested, and washed in PBS. For recombinant IL‐6 culture, cerebral vessels were cultured in DMEM+10% fetal bovine serum with either 0 or 10 ng/mL of recombinant IL‐6 (PeproTech, catalog No. 216‐16) or vehicle for 2 hours. After 2 hours of IL‐6 culture, the tissue was washed 3× with PBS, flash frozen, and used for immunoblot analysis. For anti–IL‐6 culture, cerebral vessels were cultured in DMEM+10% fetal bovine serum with either 5 μg/mL anti–IL‐6 antibody or IgG control (R&D Systems, catalog No. MAB406, clone MP5‐20F3) for 2 hours. After 2‐hour culture, tissue was washed 3× with PBS and flash frozen for use immediately for respirometry studies.
Recombinant il 6
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant IL-6 is a recombinant form of the human cytokine interleukin-6. It is a protein that plays a key role in immune response and inflammation. The core function of Recombinant IL-6 is to facilitate the study of interleukin-6 and its associated biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Phosphoramidon, by Merck Group (1 mentions)
Nα tosyl l lysine chloromethyl ketone hydrochloride tlck, by Merck Group (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Anti rb, by Cell Signaling Technology (1 mentions)
Amplex red cholesterol assay kit, by Thermo Fisher Scientific (1 mentions)
Anti mcl 1 sc 819, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p70 s6 kinase thr389, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti phospho rb s807 811, by Cell Signaling Technology (1 mentions)
Anti s6 2217, by Cell Signaling Technology (1 mentions)
Anti phospho s6 ser240 244, by Cell Signaling Technology (1 mentions)
Anti α tubulin t 5168 antibody, by Merck Group (1 mentions)
28 protocols using recombinant il 6
1
Cerebral Vessel Culture for Inflammatory Signaling
2
Evaluating IL-6 Stability with Protease Inhibitors
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To test for potential degradation of IL-6, 10 µl conditioned culture medium obtained from infection experiments was used either untreated, heat-treated (95°C/5 min), or mixed with 8 µl of different protease inhibitors (or respective controls) and then incubated with 2 µl (0.5 µg/µl) recombinant IL-6 (Peprotech, Rocky Hill) at 37°C. For inhibition of metalloproteases, phosphoramidon (Sigma-Aldrich) was used at a final concentration of 3.4 mM. Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK, Sigma-Aldrich), primarily inhibiting serine proteases, was used at a final concentration of 40 mM. Additionally, a proprietary, broad non-metalloprotease inhibitor was used (cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail, Roche, one tab dissolved in 500 µl water).
3
Comprehensive Antibody Panel for Signaling
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Anti-PARP (#9542), anti-Bcl-xL (#2762), anti-Bcl-2(#2876), anti-GAPDH (#2118), anti-cyclin D1 (#2922), anti-phospho-Rb(S807/811) (#9308), anti-Rb (#9309), anti-cleaved caspase 3 (#9661), anti-phospho-p70 S6 kinase(Thr389) (#9205), anti-S6 (#2217) and anti-phospho-S6(Ser240/244) (#5364) antibodies were from Cell Signaling Technology. Anti-phospho-STAT3(Y705) (sc-8059), anti-STAT3 (sc-7179), anti-phospho-Erk(T204) and anti-Mcl-1(sc-819) antibodies were from Santa Cruz Biotechnology. Anti-Erk (#06-182) and anti-gp130 (#06-291) antibodies were from Upstate. Anti-α-tubulin (T5168) antibody, water-soluble cholesterol (C4591) and Tyron were from Sigma Aldrich. Anti-Ki67 (#RM-9106-R7) antibody was from Thermo Scientific. Horseradish peroxidase-labeled secondary antibodies were from Jackson ImmunoResearch Laboratories. Secondary HRP-Polymer MACH3 (#M3RS31) antibody was from BioCare Medical. Secondary ImmPRESS (#MP-7401) antibody was from Vector Laboratories. Amplex Red Cholesterol assay kit (A12216) was from Molecular Probes. Recombinant IL-6 was from Pepro Tech. EUK207 was from Shirichai Orian, UCLA. MEDICA analog [HOOC-C(CH3)2-(CH2)12-C(CH3)2-COOH] was synthesized as previously described11 (link). Bortezomib was from Janssen Pharmaceutica.
4
Cell culture protocol for cancer research
5
Adipocyte Isolation and Activation
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Primary human adipocytes were prepared as described [29 (link)]. In short, aspirated adipose tissue was carefully washed with warm Hank’s medium 199 (pH 7.4, 22350029, Life Technologies, Carlsbad, CA, USA) supplemented with 5% BSA (Hyclone, Lund, Sweden) and 0.31 mg/mL of collagenase A (Roche, Basel, Switzerland) and incubated for 50 min in a shaking 37 °C water bath to allow the stromal fraction to digest. AT was thereafter filtered through the sterile 250 µm nylon filter to obtain monocellular culture and washed 4 times with pre-warmed Hank’s medium. The floating adipocyte cell layer was diluted to 1:3 in Hank’s medium supplemented with 4% BSA and metformin. The adipocyte cultures were activated for 5 h at 37 °C with recombinant IL6 (50 ng/mL, PeproTech, London, UK) in the presence of monoclonal humanized antibodies against IL6 receptor (1 mg/mL, tocilizumab (RoActemra®), Roche, Basel, Switzerland). The adipocytes were isolated by spinning the suspension through Dinonyl Phthalate oil (Alfa Aesar Chemicals, Haverhill, MA, USA). The adipocytes pellet was diluted in 350 µL RLT buffer (Qiagen) for future RNA extraction.
6
Cell Migration Assay with IL-6 and Box5
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Following serum starvation, the cells were detached with Versene and resuspended in cell media supplemented with 1% FBS; the cell concentration was then calculated using an automated cell counter (Countess™, Invitrogen). At the start of each experiment, 50,000 cells were suspended in 0.5 ml low serum media (only supplemented with 1% FBS) and added to cell culture inserts (BD Bioscience) with 8‐μm pore‐size membranes in the absence or presence of recombinant IL‐6 (Peprotech; 20 ng/ml) and Box5 (Calbiochem; 100 or 500 μM). The inserts were then placed in a 24‐well plate containing 750 μl cell media supplemented with 10% FBS, and the cells were allowed to migrate for 24 h at 37 °C in a humidified atmosphere of 5% CO2. The rate of migration was analyzed according to a protocol previously published by Broom et al. (Broom et al., 2009 ). Briefly, after fixation and staining of the migrated cells with crystal violet, the membranes were thoroughly washed in PBS to carefully remove residual staining solution. The membranes were then excised, the remaining dye was solubilized using a 10% SDS solution, and the absorbance was measured at 590 nm.
7
Neutralizing IL-6 and Modulating STAT3 in SAH
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To neutralize IL-6, intraperitoneal injection of IL-6 blocking antibody (Bio X Cell, Lebanon, NH) was administered at a concentration of 10 µg/g at 12 h post-blood injection, 48 h post-blood injection, and 72 h post-blood injection. Dosing was chosen based on prior experimental efficacy with C57BL/6 mice [14 (link)]. For the knockout mice, to confirm effect of IL-6, endogenous administration of IL-6 was performed 6 h prior to blood injection and then daily until time of euthanasia. Recombinant IL-6 (Peprotech, Inc., Rocky Hill, NJ, USA) was administered at 3.2 ng/g per day based on prior protocols [15 ]. To confirm pathway specificity, we worked with our medicinal chemist colleague to develop a custom p-STAT3 inhibitor that selectively crosses the blood brain barrier. LLL12B was administered at 5 mg/kg dosing 1, 2, and 3 days after SAH.
8
Cytokine Signaling Inhibitor Screening
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Dexketoprofen, perphenazine desloratadine and pentoxifylline (Cayman Chemicals, Ann Arbor, Michigan, USA) were dissolved in DMSO. For all the experiments, the maximal concentration of DMSO used as a control was ≤ 0.05%. Recombinant IL-6 was from Peprotech (Cranbury, NJ, USA).
9
Protein Expression and Immunoblotting
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Recombinant IL-6 was purchased from PeproTech. FAP antibody was purchased from Santa Cruz Biotech; E-cadherin, N-cadherin, Vimentin, Snail and COX-2 antibodies were purchased from Abcam; EP2 antibody was purchased from BBI; Human IL-6 mAb and mouse IL-6 mAb were purchased from R&D Systems (Minneapolis, MN, USA).
10
Th17 cell response to Mycobacterium leprae
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2 × 106 cells/ml were stimulated with or without Mycobacterium leprae sonicated antigen (MLSA; 10 μg/ml) and recombinant-IL-6 (10 ng/ml; Peprotech NJ, USA), r-TGF-β (2.0 ng/ml, Peprotech NJ, USA) and r-IL-23 (6.0 ng/ml, R&D, USA) were added with different combinations. Cultures were also stimulated with phytohemagglutinin (PHA; 5 μg/ml, Sigma, USA). Cells were stimulated in tissue culture media containing 10% FBS (Sigma, USA) and RPMI 1640 (Gibco, USA) and incubated in 5% CO2 incubator at 37 °C for 48 h. In our previous study we already performed and standardized stimulation at various time points of 6, 24 and 48 h stimulation with MLSA in leprosy patients and we observed that Th17 related genes, phenotypic characterization, cytokine release etc. in ex-vivo antigen stimulated PBMC cultures were optimum at 48 h8 (link). After these cells were used for FACS analysis and other experiments.
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