Nukleospin rna plus

Total RNA was isolated using NukleoSpin RNA Plus (Macherey-Nagel) and reverse transcribed to cDNA on a PCR System 2400 (PerkinElmer, Waltham, MA, USA), using a High Capacity cDNA Archive Kit (Applied Biosystems). Real-time PCR was performed using pre-designed TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix and run on Applied Biosystems 7500 fast software. Gene expressions of induced IL-1α (Hs00174092_m1), IL-1β (Hs01555410_m1), TNF (Hs01113624_g1), IL-6, (Hs00174131_m1), IL-8, (Hs00174103_m1) and IL-33 (Hs00369211_m1) were normalized against ACTB (Hs01060665_g1), and expressed as fold change compared to untreated control as calculated by the ΔΔCt method (ΔCt = Ct[Gene of Interest]—Ct[ACTB]; ΔΔCt = ΔCt[Treated]—ΔCt[Control]; Fold change = 2^[−ΔΔCt]).

LHC-9 cell culture medium was from Invitrogen (Carlsbad, CA, USA) and PureCol™ collagen from Inamed Biomaterials (Fremont, CA, USA). Cytokine ELISA assay for TNF-α (Human TNF-α Cytoset), IL-6 (Human IL-6 Cytoset) and CXCL8 (Human IL-8 Cytoset) were purchased from Life Technologies (Camarillo, CA, USA). Cytokine ELISA assay for IL-1β (Human IL-1β Duoset), human TLR2 antibody (MAB2616) and human TLR4 antibody (AF1478) from R&D Systems, Inc. (Minneapolis, MN, USA). NukleoSpin RNA Plus was purchased from Macherey-Nagel (Dürer, Germany) and High Capacity cDNA Archive Kit, TaqMan Universal PCR Mastermix and TaqMan Gene Expression Assay were purchased from Applied Biosystems (Foster City, CA, USA). Propidium iodide (PI) and Hoechst 33342 (Hoechst) were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). All other chemicals used were purchased from commercial sources at the highest purity available.

The cell culture medium LHC-9 and DMEM:F12, and Trypsin-EDTA, were bought from Gibco, Thermo Fisher Scientific, Waltham, MA USA. The cell culture flasks were obtained from Nunc A/S, Roskilde, Denmark, while the cell culture plates were from Corning, NY 14,831 USA. PureCol^™ collagen was from Advanced BioMatrix, Inc, CA, USA.The ELISA cytoset for CXCL8 was purchased from Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, while IL-1α and IL-1β DuoSet were from R&D Systems, Inc, Minneapolis, MN, USA. Cytotoxicity Detection Kit (lactate dehydrogenase; LDH) was obtained from Merck KGaA, Darmstadt, Germany. The RNA isolating kit; NukleoSpin RNA plus, was bought from MACHEREY-NAGEL, Duren, Germany. CH22319 and N-acetyl cystein (NAC) were bought from Merck KGaA, Darmstadt, Germany. High Capacity cDNA Archive Kit, TaqMan Universal PCR Mastermix, TaqMan Gene Expression Assays (CXCL8, Hs00174103_m1, IL-1α; Hs00174092_m1, IL-1β; Hs01555410_m1, TNF-α; Hs01113624_g1, HO-1; Hs01110250_m1, CYP1A1; Hs01054797_g1, CYP1B1; Hs02382916_s1, PAI-2 ; Hs01010736_m1, COX2; Hs00153133_m1 and GAPDH; Hs02758991_g1) were bought from Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA. Other chemicals were purchased from commercial sources at the highest purity available.