RNA extraction and cDNA synthesis were done as described in detail previously (Tartarotti et al., 2018 (link)). We used TRIzol reagent (Invitrogen) to extract total copepod RNA following the manufacturer’s instructions. We homogenised the samples and added gDNA eliminator solution (Qiagen) to eliminate potential genomic DNA contamination. The RNA extract was further purified (RNeasy Mini Kit, Qiagen; according the manufacturer’s protocol). Total RNA was measured (NanoDrop ND1000, Thermo Fisher Scientific) to assess RNA quality (mean A260/280 ratio: 2.10 ± 0.06) and run on a 1.2% agarose gel to assess RNA integrity. We quantified the RNA concentration in triplicate with a plate reader (2030 Multilabel Plate Reader Victor X4; Perkin Elmer) and the Quant‐iT RiboGreen Assay Kit (Life Technologies). Copepod RNA (450 ng) and random hexamer primers (#S0142; Thermo Scientific) were used for first‐strand complementary DNA (cDNA) synthesis. Following the manufacturer’s protocol, we synthesised cDNA using MMLV H minus reverse transcriptase (#EP0452; Thermo Scientific) with slight modifications by using less reverse transcriptase (100 U in 50‐μl reaction volume). We included no‐reverse‐transcriptase controls to test for potential genomic DNA contamination. Before quantitative polymerase chain reaction (qPCR) analysis, cDNA was frozen (−20°C).
Victor x4 2030 multilabel plate reader
Manufactured by PerkinElmer
Sourced in United Kingdom
The VICTOR X4 2030 Multilabel Plate Reader is a versatile instrument designed for high-throughput multimode detection in a variety of life science applications. It offers a wide range of detection modes, including absorbance, fluorescence, luminescence, and time-resolved fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Random hexamer primer, by Thermo Fisher Scientific (2 mentions)
Quant it ribogreen assay kit, by Thermo Fisher Scientific (2 mentions)
Mmlv h minus reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Gdna eliminator solution, by Qiagen (2 mentions)
Collagenase type 2, by Worthington (1 mentions)
Stromal cell growth medium, by Lonza (1 mentions)
Celltiter blue, by Promega (1 mentions)
Mtt assay, by Merck Group (1 mentions)
Greiss reagent, by Merck Group (1 mentions)
Facsaria 2, by BD (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Pcaggs gfp plasmid, by Takara Bio (1 mentions)
7 protocols using victor x4 2030 multilabel plate reader
1
Copepod RNA Extraction and cDNA Synthesis
2
Copepod RNA Extraction and cDNA Synthesis
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RNA extraction and cDNA synthesis were done as described in detail previously (Tartarotti et al., 2018 (link)). We used TRIzol reagent (Invitrogen) to extract total copepod RNA following the manufacturer’s instructions. We homogenized the samples and added gDNA eliminator solution (Qiagen) to eliminate potential contamination with genomic DNA. The RNA extract was further purified (RNeasy Mini Kit, Qiagen; according the manufacturer’s protocol). Total RNA was measured (NanoDrop ND1000, Thermo Fisher Scientific) to assess RNA quality (mean A260/280 ratio: 2.10 ± 0.04) and run on a 1.2% agarose gel to assess RNA integrity. We quantified the RNA concentration in triplicate with a plate reader (2030 Multilabel Plate Reader Victor X4, Perkin Elmer) and the Quant-iT RiboGreen Assay Kit (Life Technologies). Copepod RNA (450 ng) and random hexamer primers (#S0142, Thermo Fisher Scientific) were used for first-strand cDNA synthesis. cDNA was synthesized using MMLV H minus reverse transcriptase (#EP0452, Thermo Fisher Scientific) following the manufacturer’s protocol with slight modifications by using less reverse transcriptase (100 U in 50 μL reaction volume). To test for potential genomic DNA contamination, we included “no reverse transcriptase” (NRT) controls. Before quantitative polymerase chain reaction (qPCR) analysis, cDNA was frozen at −20°C.
3
LPS-induced PGE2 Secretion Assay
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Measurement of LPS-induced PGE2 production by RAW 264.7 and HS27 cells was conducted using a Cayman PGE2 Express ELISA Kit (Cayman Chemical, Ann, Arbor, MI, USA). Cells were seeded at 1.2 × 105 cells per mL of phenol-red free culture medium in 96-well plates and left to incubate overnight for 24 h. After 24 h incubation, the cells were exposed to non-toxic concentrations of CGT oil, β-bisabolol, α-bisabolol, and dexamethasone. Positive (2.5 µM (1.0 µg/mL) DEX) and negative (0.1% DMSO) controls were used and 100 ng/mL LPS was used to stimulate the cells 1 h after adding the test compounds [56 (link)]. After a further 24 h incubation, supernatants were collected from each well and PGE2 production was analyzed following the manufacturer’s instructions. Measurement of PGE2 production was by absorbance at 405 nm with a PerkinElmer VICTOR X4 2030 Multilabel plate reader (PerkinElmer, Seer Green, UK). A standard curve was constructed from dilutions of the PGE2 standard and used to determine the concentration of PGE2 produced by cells in the assay.
4
Isolation and Culture of Adventitial Fibroblasts
5
Evaluating PHNQ Cytoprotective Effects
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The effect of PHNQs on cell viability and cytoprotection against oxidative stress was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltriazolium bromide (MTT) assay (Sigma Merck, Darmstadt, Germany). Briefly, NHDF cells were seeded at a density of 104 cells/well in a 96-well microplate and incubated with increasing concentrations of PHNQs (1–100 μg mL−1) for 24 h. When indicated, after the 24 h pre-treatment with PHNQs extract, cell medium was replaced with fresh cell medium containing 5 µM Antimycin A (AMA, Thermo Scientific Chemicals, Milan, Italy), and cells were incubated for additional 2 h. Finally, 10 µL of MTT solution (5 mg mL−1 in phosphate-buffered saline solution) were added to each well and incubated for 1 h in the dark at 37 °C and 5% CO2. The incubation was stopped by the addition of 20 µL of lysis solution containing 20% (w/v) sodium dodecyl sulfate and 50% (v/v) dimethylformamide at pH 4.7. Plates were read for OD at λ = 590 nm using a Victor™ X4 2030 multilabel plate reader (PerkinElmer, Waltham, MA, USA). Cells exposed to 1% DMSO were used as control. The experiment was repeated at least three times.
6
Nitrite Quantification for Nitric Oxide
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Nitric oxide production was measured by quantifying nitrite in the cell supernatant according to previously published procedures [76 (link),77 (link),78 (link)] with some modification. RAW 264.7 cells were seeded at a density of 6 × 105 cells per mL of phenol-red free culture medium and incubated for 24 h at 37 °C in a CO2 incubator. Cells were incubated with test compounds and controls 1 h before the addition of 100 ng/mL LPS [76 (link)]. Dexamethasone (2.5 µM (1.0 µg/mL)) was used as the positive control and 0.1% DMSO as the negative control. Twenty four (24) hours after the addition of LPS, the supernatant was collected and the nitrite concentration analyzed. Greiss reagent was prepared by mixing the purchased Greiss reagent (Sigma Aldrich, Burlington, MA, USA) with 250 mL milli-Q water according to the manufacturer’s instructions. Equal volumes of prepared Greiss reagent and cell supernatant were mixed and incubated for 15 to 20 min in the dark at room temperature before absorbance readings at 550 nm were taken using a PerkinElmer VICTOR X4 2030 Multilabel plate reader (PerkinElmer, Seer Green, UK). A standard curve to quantify nitrite in cell supernatant from absorbance readings was prepared using sodium nitrite standards.
7
Insulin Secretion Assay in MIN6 Cells
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Clone B1 derived from Mouse insulinoma pancreatic 6 β-cells (MIN6 B1) were seeded at 3 × 106 cells/well in 6-well plates and co-transfected with WT or mutant FLAG-tagged RIMS2 plasmids (2 μg, Genscript) and the pCAGGS-GFP plasmid (1 μg; Clontech) via the FuGene HD transfection reagent according to the manufacturer’s protocol (Promega). GFP-expressing cells from cell pools were sorted on a BD FACSAria II (BD Biosciences) with special order research products (SORP) program. GFP-MIN6 B1 cells were seeded at 5 × 104 cells/well in 96-well plates. After 1 h of DMEM Glutamax I medium (5 mM glucose), the medium was replaced by DMEM Glutamax I medium (25 mM glucose). Concentration of insulin in the medium was determined with the Insulin Mouse ELISA Kit (Invitrogen Thermo Fisher Scientific) after 25 min of incubation. Absorbance was measured immediately at 450 nm and 550 nm by a VICTOR X4 2030 multilabel plate reader (PerkinElmer). Using Prism6 software, we determined the significance of variations among samples via a one-way ANOVA with a post hoc Tukey’s test.
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