The monocytes (4 × 105/ml) were cultured in RPMI 1640 medium supplemented with 2 mM glutamine, antibiotics and 10% fetal calf serum in the presence of 40 ng/ml recombinant human (rh) GM-CSF (rhGM-CSF was donated by Probiomed, México City, México) for M1 or 50 ng/ml of rh macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) for M2 for 5 days (the culture medium was replaced every 2 days with fresh culture medium containing rhGM-CSF or rhM-CSF). For complete polarization, M1 were stimulated with 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and 25 ng/ml IFN-γ (R&D Systems, Minneapolis, MN, USA), and M2 were cultured with 10 ng/ml IL-4 and 40 ng/ml IL-13 (R&D Systems, Minneapolis, MN, USA) for 24 h. In contrast, monocytes were cultured with 60 ng/ml rhIL-17 (R&D Systems, Minneapolis, MN, USA) for 6 days (the culture medium was replaced with fresh culture medium containing rhIL-17 every 2 days) to obtain IL-17 differentiated macrophages. For M0 macrophages (M0), monocytes were cultured only in culture medium.
Rhil 17
Manufactured by R&D Systems
Sourced in United States
RhIL-17 is a recombinant human interleukin-17 protein. Interleukin-17 is a pro-inflammatory cytokine involved in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Rhgm csf, by R&D Systems (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
M csf, by R&D Systems (1 mentions)
Il 13, by R&D Systems (1 mentions)
Rhm csf, by R&D Systems (1 mentions)
Rh il 22, by Thermo Fisher Scientific (1 mentions)
Rhifn γ, by R&D Systems (1 mentions)
Rhil 1β, by R&D Systems (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Rh gm csf, by BD (1 mentions)
Epinephrine, by PromoCell (1 mentions)
Transferrin, by PromoCell (1 mentions)
Keratinocyte growth medium 2, by PromoCell (1 mentions)
Nheks, by PromoCell (1 mentions)
Hydrocortisone, by PromoCell (1 mentions)
Hff 1, by American Type Culture Collection (1 mentions)
Hrptepic, by ScienCell (1 mentions)
6 protocols using rhil 17
1
Monocytes Differentiation into M1/M2/IL-17 Macrophages
2
Evaluating Skin Responses to Cytokine Stimuli
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Full-thickness human skin models (MatTek Corp., Ashland, MA, U.S.A.) (n = 4) were incubated in assay media (MatTek Corp.) supplemented with or without rh-IL-17 (R&D Systems, Minneapolis, MN, U.S.A.) 200 ng mL−1, rh-IL-22 (Peprotech Inc., Rocky Hill, NJ, U.S.A.) 200 ng mL−1 200, or rh-IFN-γ (R&D Systems, Minneapolis, MN, U.S.A.) 20 ng mL−1, for 2 days. On day 2, the skin models were harvested for microarray analyses. The same concentrations used for treating in vitro monolayer keratinocytes were applied for RHE, as they were proved effective in gene modulation as previously described by our group [12] (link).
3
Modulation of Human BBB Endothelial Cells
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Confluent primary cultures of human BBB ECs were either left untreated, cultured in presence of activated CD4+ T cells or treated with rh 100 U/ml IFNγ (Thermo Fisher Scientific) and rh 100 U/ml TNFα (Thermo Fisher Scientific), 100 U/ml of each alone, 1 ng/ml rh IL-1β (R&D Systems), 100 ng/ml rh IL-17 (R&D Systems), 100 ng/ml rh GM-CSF (BD Biosciences) or 40% ACM. After an overnight incubation at 37°C, cells were washed with PBS, treated with PBS-EDTA for 5 min at 37°C, dissociated with 1× trypsin (Thermo Fisher Scientific) and processed for flow cytometry analysis. For the immunocytofluorescence assays, the primary cultures of human or mouse BBB ECs were directly cultured on specialized chamber slides (Ibidi) before stimulation and analysis via confocal microscopy.
4
Cultivating human skin fibroblasts and keratinocytes
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We cultivated primary human skin fibroblast lines (HF40 and HFF-1) (n = 2 each) that were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum and, when confluent, medium was supplemented with or without recombinant human (rh)-IL-17 (R&D System, Minneapolis, MN) of 200 ng ml−1 (same IL-17 source and concentration used in prior experiments with human keratinocytes) [10] (link), [12] (link). After 24-hour incubation, fibroblasts were harvested for further analyses.
We also cultivated NHEKs obtained from PromoCell, in the Keratinocyte Growth Medium 2 supplemented with 0.004 ml/ml BPE, 0.125 ng/ml EGF, 5 ug/ml Insulin, 0.33 ug/ml Hydrocortisone, 0.39 ug/ml Epinephrine, 10 ug/ml Transferrin, and 0.06 mM Ca++ (all items purchased from PromoCell GmbH, Heidelberg, Germany). The experiment was performed in triplicate.
Once 70–80% confluent, the medium was changed with full media containing 0.06 mM Ca++, 1.2 mM Ca++, or 1.2 mM Ca++ plus 2.0% FBS, for 24 and 48 hours before harvesting for other analyses.
We also cultivated NHEKs obtained from PromoCell, in the Keratinocyte Growth Medium 2 supplemented with 0.004 ml/ml BPE, 0.125 ng/ml EGF, 5 ug/ml Insulin, 0.33 ug/ml Hydrocortisone, 0.39 ug/ml Epinephrine, 10 ug/ml Transferrin, and 0.06 mM Ca++ (all items purchased from PromoCell GmbH, Heidelberg, Germany). The experiment was performed in triplicate.
Once 70–80% confluent, the medium was changed with full media containing 0.06 mM Ca++, 1.2 mM Ca++, or 1.2 mM Ca++ plus 2.0% FBS, for 24 and 48 hours before harvesting for other analyses.
5
Evaluating IL-17 Effects on Renal Tubular Cells
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To evaluate the role IL-17 in acute and chronic renal tubular epithelial cell injury, we performed in vitro study. Human renal proximal tubular epithelial cells (HRPTEpiCs) were purchased from ScienCell Research Laboratories (ScienCell, CA, USA). HRPTEpiCs were maintained in epithelial cell medium (EpiCM, ScienCell Research Laboratories) supplemented with 2% fetal bovine serum (FBS), at 37°C with 5% CO2. HRPTEpiCs were seeded in 24 well plates at a density of 2x105 HRPTEpiCs /ml in the above described medium. After 24 hours, the medium was replaced with HRPTEpiCs culture medium with or without addition of cytokines. HRPTEpiCs were stimulated with various doses of recombinant human IL-17 (rhIL-17, R&D Systems, Inc. Minneapolis, MN)(10, 50, 100 ng/ml) for 72 h. Supernatants were harvested and stored at –80°C until analysis.
6
Immortalized Keratinocytes Respond to IL-17
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HOK16E6E7, a human immortalized oral keratinocyte cell line, was plated in 6-well plates (2 × 105/well) in keratinocyte serum-free medium (KSFM, Gibco BRL Life Technologies, Grand Island, NY, USA) containing supplemented with epidermal growth factor and calcium. After 24 hours' growth, medium with different doses of recombinant human IL-17 (rhIL-17, 0–20 ng/mL; R&D Systems, Minneapolis, MN, USA) or medium only was placed. After another 24 hours' culture, the cells were collected and kept in −70°C until the RNA isolation.
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