Hil 6

Manufactured by R&D Systems

Sourced in United States

The HIL-6 is a lab equipment product manufactured by R&D Systems. It is designed for general laboratory use. The core function of the HIL-6 is to perform tasks related to sample preparation and analysis.

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6 protocols using hil 6

TCR Retroviral Transduction in NOD Mice

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All the TCRs used for this study were chosen based on their ability to mediate T cell expansion or IL-2 secretion in response to wild type lnsB_9-23 peptide. Most of the TCRs used in this study were derived from islet-infiltrating T cells (Supplemental Table I). Generation of retroviral TCR retroviral constructs and TCR retrogenic mouse generation has been previously published (19 (link)-23 (link)). Briefly, female NOD.scid mice were injected i.p. with 150 mg/kg of 5-fluorouracil (American Pharmaceutical Partners Inc, Schaumburg, IL); bone marrow was harvested 72 hours later and cultured for 24 hours in complete DMEM supplemented with 20 % FBS, 20 ng/ml IL-3, 50 ng/ml hIL-6 and 50 ng/ml MSCF (R&D Systems, Minneapolis, MN). Bone marrow cells were spin transduced with retroviral supernatant, 6μg/ml polybrene, and freshly added cytokines for 1 hour at 37°C at 2500rpm at 24 and 48 hours, fresh media was added at 72 hours. After 96 hours, bone marrow cells were injected at about 2×10⁶ cells per recipient (~1 donor/1 recipient). Mice were test-bled for TCR reconstitution 8 weeks post-transplant for diabetes analysis, and analyzed 8 weeks post-transplant for all other experiments. The 12-4.4m1 sequence used in this study was an artificially modified version of 12-4.4 (see Supplemental Table I).

Bettini M., Blanchfield L., Castellaw A., Zhang Q., Nakayama M., Smeltzer M.P., Zhang H., Hogquist K.A., Evavold B.D, & Vignali D.A. (2014). TCR affinity and tolerance mechanisms converge to shape T cell diabetogenic potential. Journal of immunology (Baltimore, Md. : 1950), 193(2), 571-579.

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IL-6 Receptor Binding Inhibition Assay

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This neutralization assay is based on the inhibition of the binding of IL-6 to its receptors, in an ELISA-derived assay. Microtitration plates were coated with 300 ng/ml of recombinant human gp130 Fc chimera protein (R&D Systems, Lille, France) in PBS overnight at 4 °C. hIL-6 at 1,3 ng/ml and hIL-6Rα at 200 ng/ml (R&D Systems, Lille, France) were preincubated with the antibodies purified from the sera of immunized animals (at a dilution of 1:3) for two hours at 37 °C. After a saturation step with PBS containing 2% bovine serum albumin, 100 μl of the mix were added to the coated plates and incubated overnight at 4 °C. The plates were then incubated with 200 ng/ml of hIL-6 biotinylated antibody (R&D Systems, Lille, France) for two hours at 37 °C. After wash, plates were incubated with avidin-HRP (1:500) for 30 minutes at 37 °C and were revealed with 100 μl of TMB solution. The reaction was stopped with 50 μl of 1M sulfuric acid solution. Absorbance was measured at 450 nm.

Desallais L., Bouchez C., Mouhsine H., Moreau G., Ratsimandresy R., Montes M., Do H., Quintin-Colonna F, & Zagury J.F. (2016). Immunization against an IL-6 peptide induces anti-IL-6 antibodies and modulates the Delayed-Type Hypersensitivity reaction in cynomolgus monkeys. Scientific Reports, 6, 19549.

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Cytokine Profiling in Skin and Cell Cultures

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The supernatants of skin homogenate or cell culture supernatants were collected for cytokine evaluation. The samples with low yield of protein were pre-determined and excluded. Cytokine production was measured by ELISA kits of mTNF-α (BD Biosciences), hTNF-α (R&D systems), mIL-6 (R&D systems), hIL-6 (R&D systems), mIL-17 (R&D systems), mIL-33 (R&D systems) and RegIIIγ (Cloud Clone corp.) according to the manufacturer's instructions.

Wu Y., Quan Y., Liu Y., Liu K., Li H., Jiang Z., Zhang T., Lei H., Radek K.A., Li D., Wang Z., Lu J., Wang W., Ji S., Xia Z, & Lai Y. (2016). Hyperglycaemia inhibits REG3A expression to exacerbate TLR3-mediated skin inflammation in diabetes. Nature Communications, 7, 13393.

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Silencing Innate Immune Regulators

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RNA interference was performed by lipofection (RNAiMax, Life Technologies) of 5 nM Silencer Select siRNAs (Life Technologies) for 72 h. Cells were stimulated for 14 h with CpG2006 (Metabion), R848 (InvivoGen), CL075 (InvivoGen), Pam2CSK4 (EMC microcollections), human TNF or IL-1β (R&D systems), TLR7-specific RNA (5′-ACUG1CG1AG1CUU-X-UUCG1AG1CG1UCA-5, G1 is 7-deazaguanosine, X is 1,2,3-propanetriol) (14 (link)) or TLR8-specific RNA (5′-YUGCUGCCUUUG-X-GUUUCCGUCGUY-5′, Y is 1,3-propanediol, X is 1,2,3-propanetriol) (15 (link)) (Idera Pharmaceuticals). Supernatants were analyzed by ELISA for hIL-8 (BD Biosciences), hIL-6 or hTNF (R&D Systems). Efficiency of RNA interference was analyzed by SYBR Green quantitative PCR (qPCR) for MyD88, AP1M1 and AP2M1 expression normalized to HPRT.

Pelka K., Phulphagar K., Zimmermann J., Stahl R., Schmid-Burgk J.L., Schmidt T., Spille J.H., Labzin L.I., Agrawal S., Kandimalla E., Casanova J.L., Hornung V., Marshak-Rothstein A., Höning S, & Latz E. (2014). The UNC93B1 tyrosine-based motif regulates trafficking and TLR responses via separate mechanisms. Journal of immunology (Baltimore, Md. : 1950), 193(7), 3257-3261.

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Cytokine-Mediated Cell Proliferation Assay

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r hIL-6R and hIL-6 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). 2-Mercaptoethanol, Giemsa, dimethyl sulfoxide (DMSO), and MTT were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). ³H-tritiated thymidine and ATPlite kit were purchased from PerkinElmer Inc. (Waltham, MA, USA). Genomic DNA Purification Kit was purchased from Promega Corporation, (Fitchburg, WI, USA).

Wang J., Qiao C., Xiao H., Lin Z., Li Y., Zhang J., Shen B., Fu T, & Feng J. (2016). Structure-based virtual screening and characterization of a novel IL-6 antagonistic compound from synthetic compound database. Drug Design, Development and Therapy, 10, 4091-4100.

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Murine Cytokine-Induced CFU Quantification

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To assess colony-forming unit (CFU) stimulation of murine cytokines, freshly isolated mononuclear BM cells (3 × 10⁴) resuspended in IMDM and supplemented with 20 % FCS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, stem cell factor (SCF; 10 ng/ml), fms-related tyrosine kinase 3 (FLT3; 10 ng/ml), and thrombopoietin (TPO; 50 ng/ml) were mixed with methylcellulose (Methocult M3231, 2.6 %, StemCell Technologies, Vancouver, Canada) to yield a final concentration of 0.9 % methylcellulose. Additional factors were added in the following concentrations as indicated within the figure: IL-3 (20 ng/ml), GM-CSF (50 ng/ml), G-CSF (50 ng/ml), and Angptl4 (50 ng/ml). For estimation of CFU frequency after Angptl4 stimulation in vivo, 3 × 10⁴ cells were plated in methylcellulose mixed with IMDM (30 % FCS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol) including the following factors: mIL-3 (10 ng/ml), hIL-6 (10 ng/ml), mSCF (10 ng/ml), mGM-CSF (10 ng/ml), mTPO (50 ng/ml), and huEPO (2 U/ml) (all R&D Systems, Minneapolis, MN, USA).

Schumacher A., Denecke B., Braunschweig T., Stahlschmidt J., Ziegler S., Brandenburg L.O., Stope M.B., Martincuks A., Vogt M., Görtz D., Camporeale A., Poli V., Müller-Newen G., Brümmendorf T.H, & Ziegler P. (2015). Angptl4 is upregulated under inflammatory conditions in the bone marrow of mice, expands myeloid progenitors, and accelerates reconstitution of platelets after myelosuppressive therapy. Journal of Hematology & Oncology, 8, 64.

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