The day following cell seeding in 6-well plates, the cells were irradiated (10 Gy; single treatment) when they reached 70–80% confluence. At 24 h post-irradiation, cells were analyzed using a Caspase-3 apoptosis detection kit (Beyotime, Shanghai, China). Ac-DEVD-pNA (2 mM) was added to the cell lysis detection solution. After incubation for 2 h at 37 °C, absorbances (at 405 nm) were measured using an EpochTM spectrophotometer (BIO-TEK instruments Inc., Vermont, USA).
Epoch spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States, Germany, India, United Kingdom
The Epoch spectrophotometer is a high-performance, UV-visible-near-infrared spectrophotometer designed for accurate and reliable absorbance measurements. It features a monochromator-based optical system, a wide wavelength range, and a high-resolution detector.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (10 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (9 mentions)
2100 bioanalyzer, by Agilent Technologies (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Absolute blue qpcr mix, by Thermo Fisher Scientific (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (5 mentions)
Dneasy blood and tissue kit, by Qiagen (5 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (4 mentions)
Trypsin edta solution, by Merck Group (4 mentions)
Mirneasy mini kit, by Qiagen (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Take3 micro volume plate, by Agilent Technologies (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
T100 thermal cycler, by Bio-Rad (3 mentions)
Glacial acetic acid, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
230 protocols using epoch spectrophotometer
1
Caspase-3 Analysis of Irradiated Cells
2
Quantitative Real-Time PCR of OSCC Cells
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All OSCC cell lines, as well as CD44( + )-OSCC and CD44(-)-OSCC cells, total RNA was extracted with the use of the TaKaRa MiniBEST Universal RNA Extraction kit (Cat#9767, TaKaRa, Inc., Otsu, Japan). The EpochTM spectrophotometer (BIO-TEK, Vermont, USA) was used to measure the concentration and purity of the RNA. Following the manufacturer’s instructions, cDNA was synthesized via a PrimeScriptTM RT Master Mix (Perfect Real Time) kit (Cat# RR036A, TaKaRa, Inc., Otsu, Japan). For quantitative real-time PCR, we utilized the TB Green Premix Ex TaqTM II kit (Cat# RR820A, TaKaRa, Inc., Otsu, Japan). The 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) was employed for gene expression detection. The relative expression of target genes was determined via the 2-△△CT method, with GAPDH serving as an internal reference. Sangon Biotechnology Inc. (Shanghai, China) produced the primers, which are listed in Supplementary Table 1 .
3
Enzymatic Activity Quantification Protocol
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Commercially available assay kits were used to detect ACP, CTSB, and CTSD enzymatic activities in line with specific instructions. The ACP activities were measured as the absorbance with the Epoch TM spectrophotometer (BioTek Instruments) at 405 nm. For CTSB, fluorescence was detected at the emission and excitation wavelengths of 505 and 400 nm, separately, whereas 460 and 328 nm separately for CTSD using a microplate reader (PerkinElmer EnSpire).
4
Analyzing PTZ-induced Transcriptomic Changes
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Animals were crioanesthetized and their heads were immediately isolated, quickly frozen in liquid nitrogen, and stored at −80 °C 0.05, 1, 6, 12, 24, and 48 h after PTZ treatment. We pooled 20 larval heads to obtain sufficient material for RNA extraction. A total of five samples (n = 5 for each time point) were used for each group (CG, SG, and indomethacin treatment). Total RNA was extracted using TRIzol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and its concentration and quality were determined with the EpochTM spectrophotometer (BioTek, Winooski, VT, USA) and electrophoresis using agarose gels. One microgram of total RNA was reverse transcribed into cDNA using the High Capacity first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reactions without RNA were run as negative controls.
5
Antimicrobial Activity of Date Fruit Melanin
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The antimicrobial activity of date fruit melanin was investigated by basically following the method of Al-Nabulsi et al.44 (link) for minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC). Bacterial cultures (Escherichia coli 0157: H7 1934, Staphylococcus aureus ATCC 25,923, Salmonella typhimurium 02-8423, and Listeria innocua DSM 20,649) were prepared as per the method of Al-Nabulsi et al.44 (link). Date fruit melanin and reference standard of sepia melanin were dissolved in DMSO at various concentrations (1.95–1000 µg/mL) and tested. The turbidity of the 96-well plate incubated at 37 °C for 24 h was observed at 600 nm using Epoch Spectrophotometer (BioTek EPOCH 2 Microplate, Agilent, Santa Clara, USA). To determine MBC, 100 μl suspension from the MIC wells was placed on BHI agar plate and incubated for 24 to confirm its bactericidal activity.
6
Robust Total RNA Extraction and Sequencing
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For total RNA extraction, cells were pelleted at 8,500 × g during 8 min at 4 °C. After addition of RLT buffer (Qiagen) supplemented with β-mercaptoethanol, samples were sonicated on ice, RNA was then purified using RNeasy Plus Minikit (Qiagen) following the manufacturer protocol. Extracted RNA was quantified using a Biotek Epoch spectrophotometer and the quality estimated on RNA 6000 nanochips using a Bioanalyzer (Agilent). RNA extraction failed for 15 samples (all, i.e., eight, P. fraudulenta samples in medium X1; four P. fraudulenta samples in medium X2; two P. pungens samples in medium X1; and one P. australis sample in medium X1). Starting from 0.5µg of total RNA, poly-A selection, reverse transcription and library preparation was performed using the Illumina Stranded Truseq Total RNA Library Preparation Kit. One library was generated per sample, representing a total of 69 libraries. Library quality was assessed on a Bioanalyzer (Agilent) using high sensitivity DNA analysis chips and quantified using Kappa Library Quantification Kit. Paired-end sequencing was performed in 2× 150 bp on three lanes of a Hiseq3000 (Illumina). To avoid batch effects, samples were randomized for RNA extraction, library preparation, and sequencing.
7
SRB Assay for Cytotoxicity Screening
8
Quantifying Serum ADMA Levels
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Venous blood samples were obtained from all participants. Serum was separated from the blood after resting in a test tube for about 2 hours at 25℃ followed by centrifugation at 1,000×g for 15 minutes, and then stored at −20℃ until used for the ELISA.
Serum ADMA levels were quantified with an ELISA kit (Cusabio, Wuhan, China). The detection range of the kit was 7.8–500 ng/mL. All assay procedures were carried out according to the manufacturer's instructions. The absorbance values of standards and samples were obtained at 450 nm (reference wavelength 540–570 nm) using an Epoch spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).
Serum ADMA levels were quantified with an ELISA kit (Cusabio, Wuhan, China). The detection range of the kit was 7.8–500 ng/mL. All assay procedures were carried out according to the manufacturer's instructions. The absorbance values of standards and samples were obtained at 450 nm (reference wavelength 540–570 nm) using an Epoch spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).
9
Isolation and Characterization of Genomic and Plasmid DNA from Lactobacillus plantarum
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Total genomic DNA was extracted and purified from L. plantarum LL441 using the GenEluteTM Bacterial Genomic DNA kit (Sigma-Aldrich) following the manufacturer’s instructions for Gram-positive bacteria. The concentration and quality of the DNA was measured using an Epoch spectrophotometer (BioTek, Winooski, VT, United States).
The isolation of plasmid DNA from L. plantarum LL441 was performed according to the method of O’Sullivan and Klaenhammer (1993) (link) with minor modifications (the denaturation and neutralization steps were performed using the solutions provided with the commercial Plasmid Mini Kit (Qiagen, Hilden, Germany). Plasmid profiles were observed by electrophoresis in 0.75% agarose gels in 1× TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA), stained with ethidium bromide (0.5 mg mL-1), and visualized and photographed under UV light.
The isolation of plasmid DNA from L. plantarum LL441 was performed according to the method of O’Sullivan and Klaenhammer (1993) (link) with minor modifications (the denaturation and neutralization steps were performed using the solutions provided with the commercial Plasmid Mini Kit (Qiagen, Hilden, Germany). Plasmid profiles were observed by electrophoresis in 0.75% agarose gels in 1× TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA), stained with ethidium bromide (0.5 mg mL-1), and visualized and photographed under UV light.
10
Acteoside Modulates Nitric Oxide Production
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Primary rat chondrocytes were treated with 50 or 100 μM acteoside in the presence or absence of 10 ng/mL IL-1β for 24 h. Thereafter, 50 μL of the conditioned medium was reacted with 50 μL each of sulfanilamide and N-1-napthylethylenediamine dihydrochloride. Absorbance was then measured at 540 nm wavelength using a spectrophotometer (Epoch Spectrophotometer, BioTek, Winooski, VT, USA).
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