ERK phosphorylation assays were performed on HEK Flp-in hCB2 and HEK Flp-in wt cell lines. Cells were seeded at a density of 4.5 × 104 cells per well in poly-D-lysine treated clear 96-well NuncTM plates; for PTX experiments cells were plated in the presence of PTX (100 ng mL-1) or vehicle. 24–25 h later, medium was removed and replaced with basal medium (DMEM supplemented with 1 mg mL-1 BSA). Cells were equilibrated for 3 h at 37°C followed by drug stimulations carried out at 37°C for 4 min. All drugs were prepared in basal medium at 2× final concentration. An inhibitor of ERK phosphorylation, U0126 (Cell Signaling Technology, MA, United States) at 10 μM, and a pERK pathway stimulant, phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) at 100 nM, both incubated for 15 min, were utilised as reference points. At the conclusion of the drug incubation, plates were placed on ice, and immediately lysed by adding 20 μL of ice-cold lysis buffer (from AlphaLISA® Kit; details follow). Detection was performed using the AlphaLISA® SureFire® UltraTM p-ERK 1/2 (Thr202/Tyr204) Assay Kit (PerkinElmer), according to manufacturer instructions, and plates read in a CLARIOstar® reader (BMG Labtech) using standard AlphaScreen-compatible filters. Data were normalised to matched U0126 (0%) and PMA (100%) treatments, allowing compilation of data from independent experiments.
Clariostar reader
Manufactured by BMG Labtech
Sourced in Germany
The CLARIOstar is a multimode microplate reader that measures absorbance, fluorescence, and luminescence. It features advanced optics and detection systems to provide sensitive and accurate measurements across a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Mda colorimetric fluorometric assay kit, by Abcam (3 mentions)
Celltiter glo luminescence assay, by Promega (2 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (2 mentions)
Black μclear plates, by Greiner (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Alphalisa surefire ultra p erk 1 2 thr202 tyr204 assay kit, by PerkinElmer (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Ab match assembly human pap1 kit, by MBL Life Science (1 mentions)
Ab match universal kit, by MBL Life Science (1 mentions)
Mars data analysis software, by BMG Labtech (1 mentions)
Grp78, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Nodal, by R&D Systems (1 mentions)
Monolisa anti hbs plus, by Bio-Rad (1 mentions)
Monolisa anti hbc plus, by Bio-Rad (1 mentions)
Mars software, by BMG Labtech (1 mentions)
Mtt labeling reagent, by Merck Group (1 mentions)
Cellrox orange reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
34 protocols using clariostar reader
1
ERK Phosphorylation Assay in HEK Cells
2
Serum Biomarkers for Disease Monitoring
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Concentrations of Reg3α (PAP1; Ab-Match Assembly Human PAP1 kit and Ab-Match Universal kit; MBL International, Des Plaines, IL, USA), CK18F (M30 Apoptosense ELISA; Peviva AB, Sundbyberg, Sweden), and elafin (R&D Systems, Minneapolis, MN, USA) were measured from patient and healthy control serum samples by sandwich ELISA according to the manufacturers’ protocols. Serum samples with very high protein concentrations were diluted to match the protocols’ ranges of detection. Absorbance at 450 nm was measured by using the ClarioStar reader (BMG Labtech, Ortenberg, Germany). Samples were run in triplicate. Analyses were carried out with the Mars Data Analysis software (BMG Labtech).
3
CR1-Binding Protein Interaction Assay
4
Serological Markers for Hepatitis B
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Anti-HBc and anti-HBs were detected using the Monolisa Anti-HBc PLUS and the Monolisa Anti-HBs PLUS kits (Bio-Rad, Feldkirchen, Germany), respectively. Optical density was measured on a CLARIOstar reader (BMG Labtech, Ortenberg, Germany). Positivity for anti-HBs was defined as an anti-HBs level ≥ 10 mIU/mL. HBsAg was detected by HBsAg one version ULTRA ELISA (DIA.PRO Milano, Italy), which has a diagnostic specificity of 99.5% and analytical sensitivity < 0.1 WHO IU/mL. This was performed following the manufacturer’s instructions.
5
MTT Assay for Cell Viability Assessment
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For assessing the cell viability, cells were seeded in a 96-well plate (at concentrations of 2 × 103/0.32 cm2 and 4 × 103/0.32 cm2). Then, 24 h, 48 h, and 72 h after irradiation, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) labeling reagent (Sigma-Aldrich, St Louis, MO, USA) (final concentration 0.5 mg/mL) was added into each well. Two hours after incubation in a humidified atmosphere of 5% CO2 at 37 ◦C, MTT solution was removed and 150 µL of DMSO was added into each well to extract the blue MTT–formazan crystals. The absorbance measurement was performed at a 570 nm wavelength using a CLARIOstar reader (BMG LABTECH, Ortenberg, Germany). The collected data were analyzed using MARS software (BMG LABTECH, Ortenberg, Germany).
6
Oxidative Stress Measurement in Cells
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CellROX® Orange Reagent, and MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen™, Waltham, MA, USA), at concentrations of 5 μM, were used to stain 104 cells in 96-well plates and 8-well chambered coverglass. The cells were counterstained with Hoechst (Invitrogen™, Waltham, MA, USA). The stainings were performed according to manufacturer protocols. After washes with warm PBS, the fluorescence intensity was measured with a BMG Labtech (Ortenberg, Germany) CLARIOstar reader, with an excitation/emission of 545/565 nm for CellROX® Orange Reagent, and 510/580 nm for MitoSOX™ Red. The cells on the coverglass were visualized under CLSM.
7
Cytotoxicity Assay in Vero E6 Cells
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A cytotoxicity assay was performed in Vero E6 cell monolayers using the alamarBlue cell viability reagent (Thermo Fisher Scientific). Cells (2 × 104 cells/well in 100 μL) were seeded in 96-well plates. After an overnight incubation at 37 °C, 80 μL of medium was aspirated, and 20 μL of inhibitors was added in triplicate to the wells. After 1 h of incubation, 20 μL was aspirated, 150 μL of overlay was added, and the cells were incubated at 37 °C for three days. Then, 19 μL of alamarBlue reagent was added per well and incubated for 2.5 h at 37 °C. The fluorescence results were determined by excitation at 530 nm and the collection of emission at 580 nm was determined using a CLARIOstar® reader (BMG labtech, Ortenberg, Germany). The cell viability was calculated by dividing the fluorescence results of the compound-containing wells by those of the control wells (containing no compounds).
8
CyQUANT Proliferation Assay for Tumor Cells
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The CyQUANT Cell Proliferation Assay (Thermofisher Scientific) was used to determine the effect of CHI3L1 treatment or overexpression on the proliferation of tumor cells in vitro before conducting tumor experiments with these cells. Cells were plated at 1×104 cells/well in 96 well flat-bottom plates. Some plates were harvested the same day, a time when very little proliferation had occurred and considered as T0. Parallel culture plates, either control or treated, were then harvested after 48 hours or 72 hours. The assay was conducted according to the manufacturer’s instruction and acquired with a matrix-based scanning acquisition of each well on Clariostar reader (BMG Labtech).
9
In Vivo Assessment of Epithelial Barrier
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Epithelial barrier function in vivo was assessed by the leakage of orally gavaged FD4 into blood. Mice were given 600 mg/kg FD4 four hours prior to euthanasia and fluorescence in plasma at 490/525 nm was measured in a CLARIOstar Reader (BMG LABTECH). Concentration of FD4 in plasma was extrapolated by generating a standard curve, as previously described [13 (link)].
10
Combination Treatment Mitochondrial Assay
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The MTT assay was used to determine mitochondrial activity following combination treatment of MDIVI-1 and 2DG. MCF7 cells and HDQ-P1 cells were seeded at a density of 1.5 × 104 cells for well on 96-well plates, kept at 5% CO2 and 37°C and treated with increasing concentration of MDIVI-1 (from 0.1 to 10 μM) in combination with 2DG (from 0.3 to 30 mM). After 72 h, 20 μL of 5 mg/mL MTT in 1X PBS was added to each well and the plate incubated at 37°C for 4 h. Consequently medium was removed and crystals were suspended in 100 μL DMSO. Absorbance at 570 nm was recorded on a Clariostar reader (BMG Labtech, Ireland). Experiments were repeated three times on cultures from different platings; each treatment was performed in triplicate during every experiment.
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