HK1160 cells with sgRNA plasmids were grown in LB broth containing spectinomycin at 37°C for 15 h. 1.0% of cells were inoculated in 25 ml of M9 minimal medium containing sodium succinate (final 20 mM) with or without L-arabinose (final 1mM), and spectinomycin in 250 ml flasks. Six hours after the beginning of the flask culture, D-galactose (final 20 mM) was added. Cell growth was monitored by measuring optical density at 600 nm of culture broths every 3 h using an Ultrospec 8000 spectrophotometer (GE Healthcare, Sweden). The concentration of metabolites such as D-galactose and succinate in the culture was determined by high-performance liquid chromatography (RID-10A RI monitor, Shimadzu, Japan) with an Aminex HPX-87H column (300×7.8 mm, BioRad) as described previously [23 (link), 24 (link)]. After centrifugation of the cell culture broth, the supernatant was filtered by a 0.2 µm syringe filter. The column was isocratically eluted at 47°C with a flow rate of 0.5 ml/min using 0.01 N H2SO4.
Ultrospec 8000
Manufactured by GE Healthcare
Sourced in Sweden, United States
The Ultrospec 8000 is a high-performance UV-visible spectrophotometer designed for a wide range of laboratory applications. It features a precise and accurate optical system for reliable measurements across a broad wavelength range.
Automatically generated - may contain errors
Lab products found in correlation
Rid 10a ri monitor, by Shimadzu (1 mentions)
Quantstudio 1 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Real time pcr master mix, by Biofact (1 mentions)
Accupower rt premix, by Bioneer (1 mentions)
Accuprep universal rna extraction kit, by Bioneer (1 mentions)
Aprotinin, by Merck Group (1 mentions)
6 protocols using ultrospec 8000
1
Metabolite production in HK1160 cells
2
Spectrophotometric Film Transmittance
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Transmittances were determined using an Ultrospec 8000 (GE Healthcare, Chicago, IL, USA) spectrophotometer. Spectra were recorded over the range of 200–800 nm with a resolution of 1 nm. Transmittances were measured at 5 locations of each film, and the average values were reported.
3
Transcriptional Regulation of Gal Operon
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HK1060 cells carrying each crRNA plasmid were grown in LB broth containing spectinomycin at 37°C overnight as starter cultures. Next, 1.0% of cells were inoculated in 25 ml of M9 minimal medium containing sodium succinate (final 20 mM) as a carbon source and spectinomycin in 250-ml flasks. crRNAs were expressed constitutively, and gratuitous L -arabinose (final 1 mM) was added for the expression of dCas9 proteins to form the dCas9-crRNA complex in HK1060 cells carrying crRNA plasmids. To determine whether the transcription of the gal operon could be negatively regulated by the dCas9-crRNA complex, D -galactose (final 20 mM) was added as an additional carbon source 6 h after the beginning of flask cultures. Optical density at 600 nm was measured every 3 h to monitor cellular growth using an Ultrospec 8000 spectrophotometer (GE Healthcare, Uppsala, Sweden).
4
RNA Extraction and qPCR Analysis of Macrophages
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RNA was extracted from 1 × 106 BMDMs, RAW264.7 cells, and TAMs using the AccuPrep Universal RNA Extraction Kit according to the manufacturer's instructions (K-3140, Bioneer Corporation). The RNA concentration was determined by Ultrospec 8000 (GE Healthcare). Total RNA (1 μg) was reverse transcribed using AccuPower RT premix (Bioneer Corporation). For qPCR, RNA expression was analyzed on the QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific) using SFCgreen included in the 2X Real-time PCR Master Mix (DQ362-40h, BioFACT) at a concentration of 0.4 μL cDNA/sample. The primers for each of the genes are listed in Supplementary Table S1 . Quantitative gene expression data were normalized to the expression levels of β-actin.
5
Measuring Thioredoxin Reductase Activity
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The TrxR activity was measured with a commercially available kit (Sigma Aldrich). The assay is based on the catalytic reduction of 5,5′-dithiobis(2-nitrobenzoic) acid to 5-thio-2-nitrobenzoic acid by TrxR. This reduction generates a yellow colored product. Its absorbance is measurable at 412 nm by spectrophotometry. The cells were incubated 24h with or without 50 µg Au.mL−1 of GNPs before to being detached with 0.25% trypsin. The cells were pelleted by centrifugation (1000 rpm, 5 min, 4 °C) and the medium was discarded. The pellet was resuspended in a homemade lysis buffer (9% w/w sucrose; 5% v/v aprotinin (Sigma-Aldrich), in deionized water) and disrupted by a dounce homogenizer. Then, the TrxR activity was measured according to the manufacturer’s instructions. The linear increase in absorbance at 412 nm was measured during 10 min using a spectrophotometer (Ultrospec 8000; GE Healthcare, Chicago, IL, USA). The TrxR activity rate was calculated from the slope of absorbance at 412 nm versus time. Data are plotted as mean absorbance values normalized by the total protein in the sample.
6
Streptococcus mutans Biofilm Formation
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