Ng a cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataay, NJ, USA
Ng a cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataay, NJ, USA). The PCR was performed using 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. Goods have been electrophoresed on a 2 agarose gel and visualized by staining with ethidium bromide. Quantitative real-time PCR analysis Quantitative real-time PCR was performed working with a SYBR Green Master Mix and the detection of mRNA was analyzed working with an ABI StepOne Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences for the reference gene GAPDH as well as the genes of interest have 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 ). Common profile occasions employed have 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 with a melting curve evaluation. The level of target mRNA was normalized to the level of the GAPDH and compared using the manage. Information were analyzed using the DDCT technique. Sandwich enzyme-linked immunosorbent assay Cytokine levels inside the culture supernatants had been D5 Receptor manufacturer measured by a sandwich enzyme-linked immunosorbent assay (ELISA) as outlined by the manufacturer’s protocol (for TSLP, IL-1b, IL-6, IL-8, and TNF-a assay; R D Systems). Absorption from the avidin-horseradish peroxidase colour reaction was measuredat 405 nm and compared with serial dilutions of human recombinants as a typical. All samples had been performed in duplicate. Direct ELISA IL-32 levels CCR9 Storage & Stability within the culture supernatants were measured by a direct ELISA as outlined by the manufacturer’s protocol (R D Systems). Absorption with the avidin-horseradish perozidase colour reaction was measured at 405 nm and compared with serial dilutions of human recombinants as a typical. 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 96-well plates soon 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 (three 105) had been treated with BS (0.01, 0.1, and 1 mg/mL), NaCl (1 mg/mL), or Mix (three lg/mL) for two h then stimulated with IL-32 (0.1 lg/mL) for 24 h. The production of TSLP and IL-1b inside the supernatant was measured by the ELISA technique (A, B). THP-1 cells (three 106) had been treated with BS, NaCl, or Mix for 2 h then stimulated with IL-32 for five h. The mRNA expressions of TSLP were measured by real-time PCR (C). The mRNA expressions of IL-1b have been measured by real-time PCR (decrease) and RT-PCR (upper) (D). Cell viability was evaluated by an MTT assay (E). THP-1 cells were cultured in the presence of media, BS (0.01, 0.1, and 1 mg/mL), NaCl (1 mg/mL), or Mix (three lg/mL) for.