Ninety-six-well plates (Sigma, Cat number #M9410) were pre-coated with hVEGFA165 (150 ng/mL) (R&D, Cat number #293-VE), PlGF (62.5 ng/mL) (R&D, Cat number #264-PGB/CF), human PD-L1 (1 µg/mL), mouse PD-L1 (1 µg/mL), human PD-1 (1 µg/mL), and mouse PD-1 (1 µg/mL). Various amounts (from 0.1 nmol/L to 10 µmol/L) of VEGF-Grab, Ate-Grab, and atezolizumab were then added to each well via a 1/2 dilution method. Molecules bound to the coated proteins were assessed through ELISA with a horseradish peroxidase (HRP)-conjugated anti-human FC antibody (Novus, NB7449). Absorbance was determined at 450 nm using a Thermo ScientificTM VarioskanTM Flash multimode reader (Product code MIB#5250030). Data were analyzed using GraphPad Prism 7.
Varioskantm flash multimode reader
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Ireland
The VarioskanTM Flash Multimode Reader is a versatile laboratory instrument used for various types of quantitative analyses. It is designed to measure a wide range of assays, including absorbance, fluorescence, and luminescence, across multiple microplate formats. The device provides accurate and reliable data, making it a valuable tool for researchers and scientists in various fields.
Automatically generated - may contain errors
Lab products found in correlation
Trypan blue, by Merck Group (2 mentions)
Cell count reagent sf, by Nacalai Tesque (2 mentions)
96 well plate, by Greiner (2 mentions)
Hvegfa165, by R&D Systems (1 mentions)
Multiskantm fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ripa buffer, by Elpis Biotech (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Ac devd afc, by Enzo Life Sciences (1 mentions)
Xtt tests, by Promega (1 mentions)
Tnbs reagent, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Quant ittm picogreen dsdna kit, by Thermo Fisher Scientific (1 mentions)
Mtt 1 proliferation kit, by Roche (1 mentions)
Hiec 6, by American Type Culture Collection (1 mentions)
Quant ittm picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
N dodecyl β d maltoside, by Merck Group (1 mentions)
Atp measurement kit, by Thermo Fisher Scientific (1 mentions)
30 protocols using varioskantm flash multimode reader
1
Binding Affinity Evaluation of VEGF-Grab, Ate-Grab, and Atezolizumab
2
Intracellular cAMP Quantification in STC-1 Cells
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STC-1 cells were seeded at 0.5 × 106 cells/mL in a final volume of 1.25 mL per well of a 12 well plate. After 24 h, well contents were removed, and monolayers were washed with Krebs–Ringer buffer, as described above. 3-isobutyl-1-methylxanthine (IBMX) (1 mM) was added to BWSPH sample wells and control wells (positive control was 10 μM forskolin, negative control was Krebs–Ringers buffer) before a 4 h incubation. STC-1 supernatants were then aspirated off and cell lysates were collected after 10 min incubation with 0.2 mL of 0.1 M hydrochloric acid at room temperature. Lysates were then centrifuged (900× g for 5 min) and stored at −80 °C before cAMP analysis. A direct cAMP ELISA kit with a minimum detection limit of 0.39 pmol/ mL was used to measure intracellular cAMP levels, and absorbance was read at 405 nm using a microplate reader (Varioskan TM Flash Multimode Reader, Thermoscientific, Waltham, MA, USA).
3
Liver Metabolite Quantification Protocol
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Plasma aspartate aminotransferase and gamma-glutamyl transferase catalytic activity were determined with a commercial kit from Biosystems S.A., following manufacturer instructions as previously described [13 (link)] using a VarioskanTM Flash Multimode Reader (Thermo Fisher Scientific).
Quantification of free liver glucose and glycogen was performed in liver biopsies homogenized in 500 μL 2N HCl as previously described [8 (link)]. Briefly, homogenates were subjected to 100°C during an hour for glycogen digestion to glucose by acid-heat hydrolysis [24 ]. Free liver glucose and digested liver glycogen were determined using a kit from Biosystems S.A., following manufacturer instructions after neutralizing acid samples with an equal amount of 2M NaOH. Absorbance was measured at λ = 505 nm using a MultiskanTM FC Microplate Photometer (Thermo Fisher Scientific).
For liver triglyceride quantification, liver homogenates were performed according to Armour et al. [25 (link)]. In brief, liver biopsies were homogenized in lysis buffer (140 mM NaCl, 50 mM Tris and 1% Triton X-100, pH 8) and measured using a kit from Biosystems S.A., following manufacturer instructions at λ = 505 nm using a MultiskanTM FC Microplate Photometer (Thermo Fisher Scientific). For all metabolite assays intra and inter-assay coefficient of variation (CV) were less than 10%.
Quantification of free liver glucose and glycogen was performed in liver biopsies homogenized in 500 μL 2N HCl as previously described [8 (link)]. Briefly, homogenates were subjected to 100°C during an hour for glycogen digestion to glucose by acid-heat hydrolysis [24 ]. Free liver glucose and digested liver glycogen were determined using a kit from Biosystems S.A., following manufacturer instructions after neutralizing acid samples with an equal amount of 2M NaOH. Absorbance was measured at λ = 505 nm using a MultiskanTM FC Microplate Photometer (Thermo Fisher Scientific).
For liver triglyceride quantification, liver homogenates were performed according to Armour et al. [25 (link)]. In brief, liver biopsies were homogenized in lysis buffer (140 mM NaCl, 50 mM Tris and 1% Triton X-100, pH 8) and measured using a kit from Biosystems S.A., following manufacturer instructions at λ = 505 nm using a MultiskanTM FC Microplate Photometer (Thermo Fisher Scientific). For all metabolite assays intra and inter-assay coefficient of variation (CV) were less than 10%.
4
Probucol Modulates Cell Viability
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The reduction in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a purple formazan product (MTT) assay was used to determine the cell viability following probucol treatment. Briefly, the cells were treated with probucol (0, 2, 4, 8, 10, 20, 40, 80 and 100 μM) at 37°C after lipopolysaccharide (LPS, 100 ng/mL, Sigma-Aldrich)-stimulation. After 24 h, MTT (20 μL, Sigma-Aldrich) was added to each well and incubated for 4 h at 37°C. Subsequently, 150 μL of DMSO (Sigma-Aldrich) was added to each well and incubated for 10 mins at 37°C. Finally, the absorbance at 490 nm was determined on a Thermo ScientificTM VarioskanTM Flash Multimode Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
5
Mouse Plasma Renin Activity Assay
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Mouse blood samples were collected from the retro-orbital venous plexus and mixed with an anticoagulant acid-citrate-dextrose solution (ACD; 38 mM citric acid, 75 mM sodium citrate, 100 mM dextrose) [21 (link), 36 (link), 37 (link)] in Eppendorf tubes. The plasma renin activity was measured using the Renin activity kit (Abcam, Cambridgeshire, UK). The fluorescent signal was measured using a microplate reader (VarioskanTM Flash Multimode reader; Thermo Fisher Scientific, Waltham, MA, USA) at EX/EM = 540/590 nm.
6
Viability Assessment of Parasite Tachyzoites
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For experiments on parasite viability, 5 × 105 tachyzoites of each parasite species were treated for 1 h with vehicle (DMSO 0.06%) or ezetimibe (20 μm ) (37°C, 5% CO2). Thereafter, viability of tachyzoites was determined by the trypan blue (Sigma-Aldrich®) exclusion staining assay as described elsewhere (Cervantes-Valencia et al., 2019 (link)). Non-stained parasites were considered as viable. Additionally, cell viability after compound treatments was assessed by the colorimetric XTT test (Promega®) according to the manufacturer instructions. Briefly, BUVEC seeded in 96-well plate (Greiner) were incubated with DMSO, ezetimibe or ezetimibe-glucuronide (both 20 μm ) in a total volume of 50 μL for 72 h. Thereafter, 50 μL of XTT working solution was added, and samples were incubated for 4 h (37°C, 5% CO2 atmosphere). The resulting formazan product was estimated via optical density (OD) measurements at 590 nm and reference filter 620-nm wavelength using VarioskanTM Flash Multimode Reader (Thermo Scientific).
7
Caspase-3 Activity Measurement in H9C2 and SH-SY5Y Cells
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Caspase-3 activity was assessed in H2O2 or 6-OHDA-treated H9C2 and SH-SY5Y cells, respectively. The cells were washed and lysed with RIPA buffer (ELPIS-Biotech, Korea). The protein concentration was quantified, using a bicinchoninic acid (BCA) assay (Thermo Scientific, MA, USA). The cell lysates were diluted and transferred to black 384-well plates, followed by the addition of an equal volume of Ac-DEVD-AFC (Enzo, Farmingdale, NY, USA)-containing reaction buffer. After incubation, fluorescence intensity was measured at excitation and emission wavelengths of 400 and 505 nm, respectively, using a VarioskanTM Flash Multimode Reader (Thermo Scientific, MA, USA).
8
Evaluating P-gp Inhibitor Cytotoxicity and Parasite Viability
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Cell toxicity of P-gp inhibitors was assessed by colorimetric XTT tests (Promega, Madison, WI, USA) according to the manufacturer instructions. Briefly, BUVEC (n = 3) were cultured in 96-well plates (Greiner) and treated with verapamil (40 µM), valspodar (5 µM) or tariquidar (2 µM) in a total volume of 50 µl for 96 h. Thereafter, 50 μL of XTT working solution were added and the samples were incubated for 4 h (37 °C, 5% CO2 atmosphere). The resulting formazan products were estimated via optical density (OD) measurements at 590 nm and reference filter 620-nm wavelength using VarioskanTM Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). BUVEC treated with the solvent (DMSO; 0.01%) were used as negative controls.
Additionally, for experiments on parasite viability, 5 × 105 tachyzoites of each parasite species were treated for 1 h with each of the studied compounds (verapamil 40 µM, valspodar 5 µM and tariquidar 2 µM; 37 °C, 5% CO2). Viability of tachyzoites was determined by the trypan blue (Sigma-Aldrich) exclusion staining assay [60 (link)]. Non-stained parasites were considered as viable.
Additionally, for experiments on parasite viability, 5 × 105 tachyzoites of each parasite species were treated for 1 h with each of the studied compounds (verapamil 40 µM, valspodar 5 µM and tariquidar 2 µM; 37 °C, 5% CO2). Viability of tachyzoites was determined by the trypan blue (Sigma-Aldrich) exclusion staining assay [60 (link)]. Non-stained parasites were considered as viable.
9
Flavonoid Cytotoxicity Evaluation
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To evaluate the effect of flavonoids on cytotoxicity, the viabilities of the 293A cells cultured in the presence of each flavonoid were detected using a Cell Count Reagent SF (Nacalai Tesque) as described previously15 (link) with some modifications. In brief, cells were seeded at 5 × 104 cells/well into 96-well cell culture plates and pre-cultured for 24 h. After incubating for 48 h with each flavonoid at various concentrations, the culture medium was replaced with 100 μL fresh medium containing 10% (v/v) of the reagent for the WST-8 assay. After 1 h of further incubation, the absorbance at 450 nm derived from WST-8-formazan in culture medium was measured using a VarioskanTM Flash Multimode Reader (Thermo Fisher Scientific, Yokohama, Japan).
10
Fluorescent Protein Immobilization Screening
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For this, 100 mg of support were incubated with 900 µL of cell extract at 0.1 mg/mL (in 25 mM sodium phosphate buffer at pH 7.5) for 1 h at room temperature with orbital shaking, unless otherwise specified. The immobilization course was followed by measuring the fluorescence in the supernatant (30 µL) using NUNCTM 384-well black plates and VarioskanTM Flash Multimode Reader (Thermo Scientific). Afterwards, the suspension was filtered (for graphene-based supports, TALON®, AG-Co2+/S, Purolite® and gDNA-coated agarose) or centrifuged (in the case of UCNP). In all cases, the immobilization yield (Ψ) was calculated as follows:
where the FPinitial is protein concentration of the fluorescent protein solution offered to the carrier, while FPsupernatant is the concentration of fluorescent protein that remains in the supernatant after the immobilization time.
The immobilization of sGFP on Purolite® was carried out in presence of 10% Triton X-100 to avoid hydrophobic interactions. After protein immobilization, 0.1 g of beads was rinsed with 1mL of a 10% Triton X-100 solution for 5 min with orbital shaking.
where the FPinitial is protein concentration of the fluorescent protein solution offered to the carrier, while FPsupernatant is the concentration of fluorescent protein that remains in the supernatant after the immobilization time.
The immobilization of sGFP on Purolite® was carried out in presence of 10% Triton X-100 to avoid hydrophobic interactions. After protein immobilization, 0.1 g of beads was rinsed with 1mL of a 10% Triton X-100 solution for 5 min with orbital shaking.
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