Bioruptor ucd 250

We used ChIP-IT Express (Active Motif, Carlsbad, CA) to perform ChIP assays. Briefly, chromatin complexes isolated from cells fixed with 1% formaldehyde were sonicated (Bioruptor UCD-250, Cosmobio, Tokyo, Japan) and immunoprecipitated by control IgG or RNA polymerase II antibody. The IL-8 promoter region contained in the immunoprecipitated samples were quantitated on a real-time PCR machine. The primers and probe set for IL-8 promoter region was as follows; Forward primer, 5′-CATCAGTTGCAAATCGTGGA-3′, Reverse primer, 5′-AGAACTTATGCACCCTCATCTTTT-3′, probe, Roche Universal ProbeLibrary Probe #6 (Roche applied Science).

The cells were washed with phosphate-buffered saline (PBS) and resuspended in 50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Nonidet P-40 (radioimmune precipitation assay buffer), and protease inhibitors. After ultrasonication using Bioruptor (UCD-250, Cosmo Bio) for 12.5 min (10 s on, 20 s off) at the middle level of output (250 W) at 4 °C, the soluble cellular extracts were recovered after centrifugation for 10 min at 16,000 × g. The protein concentration of each sample was determined using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific), and cell extracts were subjected to WB analysis. The blots were probed with the primary antibody followed by a horseradish peroxidase-conjugated secondary antibody. The immune complexes were visualized using Pierce^TM Western blotting Substrate Plus (Thermo Fisher Scientific). WB results were documented and quantified using ImageQuant LAS 4000 mini and Amersham Biosciences Imager 600 (GE Healthcare).

Cytosolic extracts were prepared as previously described^{56 (link)}. Briefly, 293TT cells were transfected with the recombinant expression plasmids pΔ102AIF, p16E6, and p6E6. Two days after transfection, the cells were washed twice with PBS and harvested with a cell scraper. The cells were suspended in fractionation buffer (250 mM sucrose, 20 mM HEPES, pH 7.3, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and proteinase inhibitor cocktail) and sonicated (Bioruptor UCD-250; Cosmo Bio, Tokyo, Japan) for 15 min with intervals of 30 s on and 30 s off at 4 °C. The cell lysates were centrifuged at 20,000×g for 30 min at 4 °C, and the supernatant, as a cytosolic extract, was stored at –80 °C until use. The cytosolic extracts from normal cells were used as a control.

ChIP for small cell numbers was performed with a Low Cell ChIP-Seq Kit (Active Motif) according to the manufacturer’s manual (version A3) with some modifications. The blastocysts were freed from the zona pellucida by using pronase before crosslinking with formaldehyde. After crosslink-quenching, the sample was sonicated to shear chromatin using a Bioruptor UCD-250 (Cosmo Bio) for 30 × 30 s with 30-s pauses in ice-water. The sample was centrifuged for 2 min at 18,000 × g and the supernatant (200 μL) was transferred to a new tube. The 200-µL sample of sheared chromatin was divided into a 10-µL aliquot as “input” and the rest (190 µL). The latter aliquot was processed for ChIP using 3 µg anti-H3K4me3 antibody (pAb-003-050, Diagenode) as indicated in the user manual. The input and ChIPed DNA was decrosslinked, purified, and resuspended in 40-µL low-EDTA TE buffer. The DNA samples were processed for library preparation for next-generation sequencing by using a Next Gen DNA Library Kit (Active Motif) following the manufacturer’s manual. The specific enrichment of H3K4me3 in the ChIP-seq libraries was validated by quantitative PCR for positive (1st exon–intron boundary of GAPDH) and negative (2nd exon of MB) regions.

To quantify the betanin content and antioxidant activity of each sample, 100 mg of stem was ground to a fine powder in liquid nitrogen and transferred to a 1.5-mL tube to which 1.0 mL of ultrapure water was added. Extraction was then facilitated by vortexing and sonicating the sample five times for 30 sec using a BIORUPTOR UCD-250 (Cosmo Bio). The resultant solution was centrifuged at 4°C and 14,000 × g for 20 min, and the supernatant was analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) or using the Folin-Ciocalteu method.

Samples were obtained from cells grown on LB plates at 37°C for 10 h. The populations were harvested by centrifugation at 21,600×g for 1 min at 4°C. The harvested cells were washed three times with 0.1 M Tris–HCl (pH 7.0) buffer. The washed cells were re-suspended and sonicated for 20 min (20 s treatment was repeated with 20-s intervals) at 4°C with a Bioruptor UCD-250 (Cosmo Bio Co. Ltd.; Tokyo, Japan). The disrupted cells were centrifuged at 21,600×g for 10 min at 4°C to remove cell debris and then subjected to ultracentrifugation at 166,000×g for 60 min at 4°C to remove the insoluble fraction. The soluble fraction was used in SDS-PAGE analysis using the NuPAGE SDS-PAGE Gel system (Invitrogen). Proteins were separated by electrophoresis using 4–12% Bis–Tris NuPAGE gels with MES running buffer, and then stained with Coomassie Brilliant Blue. Protein concentration in the soluble fraction was determined using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific Inc.; Waltham, MA, USA). The molecular weight marker was Novex^™ Sharp Unstained Protein Standard (Invitrogen).