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Hil 4

Manufactured by R&D Systems
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The HIL-4 is a laboratory equipment product from R&D Systems. It is designed for conducting research and analysis tasks. The core function of the HIL-4 is to provide a controlled environment for testing and experimentation, but a more detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Phorbol myristate acetate (pma), by Merck Group (2 mentions) Hil 13, by R&D Systems (2 mentions) Htgf β, by Thermo Fisher Scientific (2 mentions) Ficoll paque plus, by GE Healthcare (2 mentions) 96 well round bottom plate, by Corning (2 mentions) Soluble anti hcd3, by Thermo Fisher Scientific (2 mentions) Anti hcd28, by Thermo Fisher Scientific (2 mentions) Hil 1β, by R&D Systems (2 mentions) Anti hifnγ, by R&D Systems (2 mentions) Anti hcd3 cd28 conjugated dynabeads, by Thermo Fisher Scientific (2 mentions) Ficoll hypaque, by GE Healthcare (1 mentions) Anti human cd3 ucht1, by BioLegend (1 mentions) Easysep human cd14 positive selection kit, by STEMCELL (1 mentions) Na ve cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions) Lipopolysaccharide lps, by Merck Group (1 mentions) Htnf α, by Merck Group (1 mentions) Elispot assay kit, by R&D Systems (1 mentions) Ionomycin, by Bio-Techne (1 mentions) Eb h209, by Thermo Fisher Scientific (1 mentions) Hgm csf, by Merck Group (1 mentions)

7 protocols using hil 4

1

Generation of Human Monocyte-Derived Dendritic Cells

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Human mo-DCs were prepared as described previously [90 (link)]. Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor blood by Ficoll-Hypaque (GE Healthcare) density centrifugation. CD14+ cells (purity > 95%) were isolated using the EasySep™ Human CD14 Positive Selection Kit (STEMCELL) and cultured in RPMI-1640 medium supplemented with penicillin/streptomycin, 10% FBS, recombinant hGM-CSF (100 ng/ml) and hIL-4 (100 ng/ml; both from R&D) for 6 days. Human mo-DCs were treated with 10 µM SB203580 and HDM for 24 h, washed extensively and cocultured with human blood naïve CD4+ T cells isolated using the Naïve CD4+ T Cell Isolation Kit II (Miltenyi Biotec) at a ratio of 1:10. After 7 days of coculture, live T cells were purified and stimulated with plate-bound anti-human CD3 (UCHT1; BioLegend) for 5 h and then harvested for mRNA analysis. PBMCs were collected from allergic rhinitis (AR) patients and stimulated with the p38 inhibitor SB203580 or DMSO (vehicle) for 8 h, with GolgiStop added to the culture medium for the last 5 h. Cells were harvested, and IL-12p40 (C8.6, eBioscience) expression in DCs was detected by ICS. This study was approved by the Ethics Committee of the Eye & ENT Hospital of Fudan University (2017-0301). Informed consent was obtained from all volunteers.
Han M., Ma J., Ouyang S., Wang Y., Zheng T., Lu P., Zheng Z., Zhao W., Li H., Wu Y., Zhang B., Hu R., Otsu K., Liu X., Wan Y., Li H, & Huang G. (2022). The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation. Cellular and Molecular Immunology, 19(7), 805-819.
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2

Polarization of THP-1 Macrophages

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THP-1 cells (2 × 105 cells) were added into the six well plates per well with 50 ng/mL of 12-O-tetradecanoylphorbol-l3-acetate (PMA, Sigma, Cat#P1585) containing the 2 mL medium for 48 h. Activated THP-1 cells were differentiated to M1 or M2 macrophage by treating with 100 ng/mL of LPS (lipopolysaccharides) (Sigma, Cat#L2630) and 20 ng/mL of hIFN-ɤ (R&D systems, Cat#285-IF-100) in com RPMI or treating with 20 ng/mL of hIL-4 (R&D systems, #204-IL-010) and 20 ng/mL of hIL-13 (R&D systems, Cat#213-ILB-005) in com RPMI for 2 days.
Pahk K.J., Shin C.H., Bae I.Y., Yang Y., Kim S.H., Pahk K., Kim H, & Oh S.J. (2019). Boiling Histotripsy-induced Partial Mechanical Ablation Modulates Tumour Microenvironment by Promoting Immunogenic Cell Death of Cancers. Scientific Reports, 9, 9050.
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3

Induction of Human Regulatory T Cells

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Peripheral blood was obtained from healthy donors (n=7) with informed consent (WSBTS-12-10). Mononuclear cells were purified by Ficoll-Paque Plus (GE Healthcare), followed by CD4+ T cell isolation using AutoMacs. The cells were cultured (1×106/ml) for 5 days in round-bottom 96 well plates (Costar) with mitomycin C-treated autologous APC (1×106/ml) together with soluble anti-hCD3 (1.5 μg/ml, Invitrogen) and anti-hCD28 (1 μg/ml, Invitrogen) plus hIL-4 (10 ng/ml, R&D System), hTGFβ (2.5 ng/ml, eBioscience), hIL-1β (10 ng/ml, R&D System), anti-hIFNγ (10 ng/ml, R&D System) in the presence of NOC-18. In some experiments, CD4+ T cells were cultured with anti-hCD3/CD28-conjugated Dynabeads (4×107/ml, Invitrogen) instead of APC, with similar results. At the end of the culture, supernatants were harvested for ELISA and the cells were stained for intracellular cytokines.
Niedbala W., Besnard A.G., Nascimento D.C., Donate P.B., Sonego F., Yip E., Guabiraba R., Chang H.D., Fukada S.Y., Salmond R.J., Schmitt E., Bopp T., Ryffel B, & Liew F.Y. (2014). Nitric oxide enhances Th9 cell differentiation and airway inflammation. Nature communications, 5, 4575.
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4

Dendritic Cell Differentiation and Transfection

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About 4x106 THP-1 cells were seeded in T75 flasks and differentiated towards immature dendritic cells (iDC) over a 5-day culture in 20 mL serum-supplemented RPMI 1640 in the presence of 100 ng/mL hIL4 (R&D Systems, 204-IL-020/CF) and 100 ng/mL hGM-CSF (Sigma-Aldrich, #GF304). To allow full maturation towards mature dendritic cells (mDC), iDC were collected via centrifugation, resuspended in serum free RPMI 1640 media supplemented with 200ng/mL hIL4, 100ng/mL hGM-CSF, 20ng/mL hTNFα (Sigma-Aldrich, #GF314), and 200ng/mL ionomycin (Tocris Bioscience, 2092/1), and plated at density 10,000/well in the 96-well plates provided with the ELISPOT assay kit R&D Systems, #EL485, Minneapolis, MN. Cells were kept in culture for 1 day to allow differentiation towards mDC, before beginning the transfection with MessengerMax-formulated mRNA to induce expression of the E6-E7 fusion protein in the vaccine. The day following the transfection, human antigen specific E711-20 T cells (Charles River Laboratories, #ASTC-1099) were added to the 96-well plate at density 20,000 cells/well. ASTC and mDC cells were kept in coculture for an overnight. The plates were processed as per manufacturer (R&D Systems, #EL485)’s instructions and read under the CTL Immunospot Analyzer (ImmunoSpot®). CD209 monoclonal antibody eB-h209 (eBioscience™) was used to characterize DC differentiation via Flow Cytometry.
Zhou K., Yuzhakov O., Behloul N., Wang D., Bhagat L., Chu D., Zhang X., Cheng X., Fan L., Huang X, & Mirabella T. (2023). HPV16 E6/E7 -based mRNA vaccine is therapeutic in mice bearing aggressive HPV-positive lesions. Frontiers in Immunology, 14, 1213285.
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5

Preparation of Chemical Compounds for Cell Culture

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Acriflavine (Sigma-Aldrich, MO, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and stored at room temperature. TGF-β1 (R & D Systems Europe Ltd) was dissolved in 4mM HCL containing 0.1% BSA and stored at −20°C. Cobalt chloride (CoCl2) (Sigma) was dissolved in distilled water (100 mg/mL) and stored at room temperature. PMA, LPS, Curcumin, Metformin and Chloroquine were obtained from Sigma, hIL4 and hIL13 from R&D Systems.
Bulle A., Dekervel J., Deschuttere L., Nittner D., Van Cutsem E., Verslype C, & van Pelt J. (2020). Anti-Cancer Activity of Acriflavine as Metabolic Inhibitor of OXPHOS in Pancreas Cancer Xenografts. OncoTargets and therapy, 13, 6907-6916.
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6

Differentiation of Monocytes to Immature DCs

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Human immature DCs were differentiated from Monocytes as previously described19 (link)21 (link). All experimental protocols were approved by the Institutional Review Boards of Radboud University Medical Center (Netherlands) and Vimercate Hospital (Italy). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats (Sanquin) obtained from healthy volunteers after written informed consent and all methods were carried out in accordance with the approved guidelines. Briefly, monocytes were purified from Peripheral Blood Mononuclear Cells (PBMCs) using the anti-human CD14 MicroBeads (Miltenyi Biotech, Calderara di Reno, Italy). Monocytes were differentiated into iMDDCs with hIL-4 and hGM-CSF (both 20 ng/ml; R&D Systems, Minneapolis, USA) for 6 days.
Berzi A., Ordanini S., Joosten B., Trabattoni D., Cambi A., Bernardi A, & Clerici M. (2016). Pseudo-Mannosylated DC-SIGN Ligands as Immunomodulants. Scientific Reports, 6, 35373.
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7

Induction of Human Regulatory T Cells

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Peripheral blood was obtained from healthy donors (n=7) with informed consent (WSBTS-12-10). Mononuclear cells were purified by Ficoll-Paque Plus (GE Healthcare), followed by CD4+ T cell isolation using AutoMacs. The cells were cultured (1×106/ml) for 5 days in round-bottom 96 well plates (Costar) with mitomycin C-treated autologous APC (1×106/ml) together with soluble anti-hCD3 (1.5 μg/ml, Invitrogen) and anti-hCD28 (1 μg/ml, Invitrogen) plus hIL-4 (10 ng/ml, R&D System), hTGFβ (2.5 ng/ml, eBioscience), hIL-1β (10 ng/ml, R&D System), anti-hIFNγ (10 ng/ml, R&D System) in the presence of NOC-18. In some experiments, CD4+ T cells were cultured with anti-hCD3/CD28-conjugated Dynabeads (4×107/ml, Invitrogen) instead of APC, with similar results. At the end of the culture, supernatants were harvested for ELISA and the cells were stained for intracellular cytokines.
Niedbala W., Besnard A.G., Nascimento D.C., Donate P.B., Sonego F., Yip E., Guabiraba R., Chang H.D., Fukada S.Y., Salmond R.J., Schmitt E., Bopp T., Ryffel B, & Liew F.Y. (2014). Nitric oxide enhances Th9 cell differentiation and airway inflammation. Nature communications, 5, 4575.
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