HIMECs were seeded into 100 mm cell culture plates (2 × 106 cells/well) and stabilized for 24–36 h. HIMECs were stimulated with 100 ng/mL human IL-17C (eBioscience, San Diego, CA, USA) for 3 h, and harvested after incubation. Total RNA was isolated using the RNeasy plus mini kit (Qiagen, MD, USA). RNA samples (1000 ng) were reversely transcribed to complementary DNA (cDNA) using the RT & GO mastermix (MPbio, Santa Ana, CA, USA) and oligo (dT) primers (ELPIS-Biotech, Daejeon, South Korea). Microarray was performed in the RT2 Profiler PCR Array plate PAHS-169ZA (Qiagen), for screening the genes involved in human Crohn’s disease. Microarray and quantitative real-time PCR (qPCR) were performed using the 96-well plate format in Thermal cycler TP800 (TAKARA, Otsu, Japan).
Human il 17c
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human IL-17C is a recombinant protein that represents the mature form of the human interleukin-17C cytokine. IL-17C is a member of the IL-17 family of cytokines and is involved in inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse te2000 u microscope, by Nikon (2 mentions)
Thermal cycler tp800, by Takara Bio (1 mentions)
Rt go mastermix, by MP Biomedicals (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Oligo dt primers, by Elpis Biotech (1 mentions)
Rhodamine phalloidin, by Cytoskeleton (1 mentions)
Lab tek, by Thermo Fisher Scientific (1 mentions)
Formalin, by Merck Group (1 mentions)
Axioskop fl, by Zeiss (1 mentions)
Metamorph microscopy automation and image analysis software, by Molecular Devices (1 mentions)
Triton x 100, by Daejung Chemicals (1 mentions)
Matrigel, by BD (1 mentions)
Matrigel, by Nikon (1 mentions)
Endothelial cell growth media, by Lonza (1 mentions)
Multi analyte elisarray kit, by Qiagen (1 mentions)
5 protocols using human il 17c
1
Crohn's Disease Gene Expression Analysis
2
Fluorescent Imaging of Cytoskeleton in IL-17C Treated HIMECs
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HIMECs were seeded at a density of 1 × 103 cells/well on fibronectin-coated glass chamber slides (LabTek, Thermo Fisher Scientific). The cells were stabilized at 37 °C for 40 h, and treated with 100 ng/mL human IL-17C (eBioscience) for 15 min. The staining was performed as previously described [22 (link)]. Briefly, the cells were then washed with PBS, fixed with 10% formalin (Sigma-Aldrich) for 15 min, and permeabilized with Triton X-100 (0.1%, Daejung, Gyeonggi-do, South Korea) for 5 min. After permeabilization, the cells were washed twice and stained for 15 min in the dark with rhodamine–phalloidin (100 nM/well, Cytoskeleton, Denver, CO, USA). The cells were then washed three times, mounted on slides with 50% glycerol, and examined with fluorescence microscopy Axioskop FL (Carl Zeiss Meditec, Inc., Dublin, CA, USA), using Metamorph Microscopy Automation and Image Analysis Software (Molecular devices, Sunnyvale, CA, USA).
3
Matrigel Tube Formation Assay
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For this assay, 400 μL of matrigel (BD Biosciences, San Jose, CA, USA) was added to each well of the 24-well plates and incubated at 37 °C for 3 h. After polymerization, 1 × 105 HIMECs were seeded into each well and incubated with endothelial cell growth media (Lonza) with/without 100 ng/mL human IL-17C (eBioscience). After 4 h, tube formation was observed and photographed using the Nikon ECLIPSE TE 2000-U microscope (Nikon). To explore the effect of DLD-1 CM on angiogenesis, HIMECs were plated on matrigel, supplemented with normal and IL-17C-induced CM from DLD-1 cells for 6 h, and photographed by a Nikon ECLIPSE TE 2000-U microscope (Nikon). The total tube length in each well was measured using ImageJ v.1.47 software.
4
IL-17C Induced VEGF Secretion
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NCM460 and CRC cells (Caco-2, HT-29, DLD-1, and HCT116), and xenograft tumors were stimulated with 100 ng/mL human IL-17C (eBioscience) for 8 h, and the cultured supernatants were collected to detect secreted proteins using the human VEGF ELISA Duo Kits (R&D Systems, Minneapolis, MN, USA). To investigate IL-17C-related cytokines in DLD-1 cells, ELISA was conducted using the human inflammatory cytokines and chemokines multi-analyte ELISArray Kit (Qiagen, MEH-004A), following the manufacturer’s instructions. Sample absorbance values were determined at 450 nm using 540 nm as a reference wavelength, and the standard curve was calculated using a four-parameter logistics curve-fitting algorithm.
5
Wound Healing Assay with IL-17C
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HIMECs were seeded at a density of 1 × 105 cells/well into a fibronectin-coated 24-well plate and stabilized for 48 h. A scratched wound was created with a plastic pipette tip and washed with phosphate-buffered saline (PBS) for cell debris removal. The cells were then incubated with medium containing 100 ng/mL human IL-17C (eBioscience) for 24 h. DLD-1 cells were seeded at a density of 1 × 106 cells into a 24-well plate, stabilized for 48 h, and supplemented with IL-17C for 24 h. Conditioned medium (CM) was obtained from NCM460 and DLD-1 cells incubated with/without IL-17C for 8 h. HIMECs were wounded and supplemented with each CM for 12 h. Cells were pretreated with Ki8751 (4.7 g/mL; Calbiochem, Gibbstown, NJ, USA) for 30 min, and then, CM with/without IL-17C were added for additional 8 h. HIMECs were wounded and supplemented with each CM for 20 h. Cell migration and wound closure were calculated as the percentage of the total wound area at 24 h to the initial wound area. Three different areas from each group were photographed using the Nikon ECLIPSE TE 2000-U microscope (Nikon, Tokyo, Japan). The wound area was analyzed using ImageJ v1.47 software.
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