Th ten fetal bovine serum and 1 penicillin-streptomycin at 37 within a humidified atmosphere in the presence of 5 CO2. Reverse transcription PCR analysis We isolated the total RNA from THP-1 cells applying an easyBLUETM RNA extraction kit (iNtRON Biotechnology, Sungnam, Republic of Korea) in accordance with the manufacturer’s specification. Total RNA (two.five lg/mL) was heated at 65 for 10 min then chilled on ice. Each sample was reversetranscribed to cDNA for 90 min at 37 making use of a cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataay, NJ, USA). The PCR was performed together with the following primer for human IL-1b (50 -CCG GAT CCA TGG CAC CTG TAC GAT CA-30 ; 50 -GGG GTA CCT TAG GAA GAC ACA AAT TG30 ); IL-8 (50 -CGA TGT CAG TGC ATA AAG ACA-30 ; 50 -TGA ATT CTC AGC CCT CTT CAA AAA-30 ); IL-32 (50 TGA CAT GAA GAA GCT GAA GGC-30 ; 50 -CAT GAC CTT GTC ACA AAA GCT C-30 ); and GAPDH (50 -CAA AAG GGT CAT CAT CTC TG-30 ; 50 -CCT GCT TCA CCA CCT TCT TG-30 ). The annealing temperature was 62 for IL-1b, IL-8, IL-32, and GAPDH. Products were electrophoresed on a 2 agarose gel and visualized by staining with ethidium bromide. Quantitative real-time PCR evaluation Quantitative real-time PCR was performed using a SYBR Green Master Mix along with the detection of mRNA was analyzed RIPK3 Protein Source employing an ABI StepOne Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences for the reference gene GAPDH and also the genes of interest had been as follows: GAPDH (50 -TCG ACA GTC AGC CGC ATC TTC TTT-30 ; 50 -ACC AAA TCC GTT GAC TCC GAC CTT-30 ); human TSLP (50 -CCC AGG CTA TTC GGA AAC TCA G30 ; 50 -CGC CAC AAT CCT TGT AAT TGT G-30 ); human IL-1b (50 -AAA CAG ATG AAG TGC TCC TT-30 ; 50 -TGG AGA ACA CCA CTT GTT GC-30 ); human CD11b (50 -ACG TAA ATG GGA CAA GCT G-30 ; 50 -GAT CCT GAG GTTTHE EFFECTS OF BAMBOO SALT ON ARCCG TGA AA-30 ); human CD14 (50 -ACT TGC ACT TTC CAG CTT GC-30 ; 50 -GCC CAG TCC AGG ATT GTC AG30 ). Typical profile occasions used had been the initial step, 95 for ten min followed by a second step at 95 for 15 s and 60 for 30 s for 40 cycles having a melting curve evaluation. The level of target mRNA was normalized to the degree of the GAPDH and compared with the manage. Data had been analyzed making use of the DDCT approach. Sandwich enzyme-linked immunosorbent assay Cytokine levels within the culture supernatants have been measured by a sandwich enzyme-linked immunosorbent assay (ELISA) based on the manufacturer’s protocol (for TSLP, IL-1b, IL-6, IL-8, and TNF-a assay; R D Systems). Absorption in the avidin-horseradish peroxidase color reaction was measuredat 405 nm and compared with serial dilutions of human recombinants as a common. All samples were performed in duplicate. Direct ELISA IL-32 levels inside the culture supernatants had been measured by a direct ELISA in accordance with the manufacturer’s protocol (R D Systems). Absorption from the avidin-horseradish perozidase color reaction was measured at 405 nm and compared with serial dilutions of human recombinants as a common. All samples have been performed in duplicate. MTT assay Cell viability was determined employing an MTT assay. Briefly, 100 lL of cell suspension (1 ?104 cells) was cultured in Basigin/CD147 Protein Biological Activity 96-well plates after pretreatment by each concentration of BS,FIG. 1. BS inhibited the IL-32-induced production and mRNA expression of TSLP and IL-1b. THP-1 cells (3 ?105) were treated with BS (0.01, 0.1, and 1 mg/mL), NaCl (1 mg/mL), or Mix (three lg/mL) for 2 h and then stimulated with IL-32 (0.1 lg/mL) for 24 h. The production of TSLP and IL-1b within the superna.
