Peptide therapeutics for autoimmune and inflammatory diseases
A technology for autoimmune diseases and inflammatory diseases, applied in allergic diseases, anti-inflammatory agents, non-central analgesics, etc.
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Embodiment 1
[0090] Example 1: Materials and Methods
Embodiment 1-1
[0091] Example 1-1: Peptide synthesis, reagents and cell line optimization
[0092] Peptides were synthesized by Biostem (Ansan, Korea) with 97.09% (MIP1), 97.17% (MIP2) and 95.1% (MIP3) purities, respectively, as determined by reversed-phase high performance liquid chromatography (HPLC; Shimadzu Prominence). All peptides were detected at 220 nm using a Shiseido Capcell pak C18 column (4.6 mm x 50 mm) with a gradient of 10%-60% acetonitrile in 0.1% trifluoroacetic acid (TFA)-water at a flow rate of 1 mL / min. The molecular weights of MIP1, MIP2 and MIP3 were measured by Shimadzu LCMS-2020 and were 2182.6 Da, 3710.5 Da and 3680.5 Da, respectively. LPS (E. coli 0111:B4) and adenosine triphosphate (ATP) were purchased from Sigma-Aldrich. PAM 3 CSK 4 (TLR1 / 2), Poly(I:C)(TLR3), Imiquimod (IMQ; R837, TLR7), R848 (TLR7 / 8) and CpG-ODN (TLR9) were purchased from Thermo Fisher Scientific, Inc., and FSL-1 (TLR2 / 6) were purchased from InvivoGen (San Diego, CA, USA).
[0093] Put HEK-B...
Embodiment 1-2
[0094] Example 1-2: Cell Viability Assay
[0095] Put HEK-Blue TM hTLR4 cells at 5 × 10 4 Density distribution of cells / well, RAW264.7 cells and THP-1 cells were distributed at 2 × 10 5 Density assignment of cells / well. All cells were grown overnight in 96-well plates (BD Biosciences, San Jose, CA, USA). Cell viability was determined using the colorimetric 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylmethanezan (MTT) assay (Sigma-Aldrich) and / or MTS (3-(4 , 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole) (Promega Madison, WI, USA) to assay, performed as previously described (Kwon, H.K. et al., Toxicological sciences: an official journal of the Society of Toxicology 148, 204-219 (2015)).
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