The plasma concentration of myoglobin was determined in 20 μL of plasma using the rat myoglobin ELISA kit (Life Diagnostic Inc., West Chester, PA) according to the manufacturer’s instructions. Data were collected spectrophotometrically using a standard 96-well plate reader at a wavelength of 450 nm (Sunrise™ reader, Tecan Group Ltd., Männedorf, Switzerland).
Sunrise reader
Manufactured by Tecan
Sourced in Switzerland, Austria, Germany
The Sunrise reader is a high-performance microplate absorbance reader designed for a wide range of applications in life science research and clinical diagnostics. It provides accurate and reliable measurements of optical density in 96-well or 384-well microplates. The Sunrise reader is a compact and user-friendly instrument that delivers consistent and reproducible results.
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Lab products found in correlation
Superoxide dismutase assay kit, by Cayman Chemical (2 mentions)
Goat anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Magellan v 5, by Tecan (2 mentions)
Bio plex 200, by Bio-Rad (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Bio-Rad (1 mentions)
Streptavidin coated plates, by Roche (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Transam nf κb p65 protein assay, by Active Motif (1 mentions)
Cobas e 411 analyzer, by Roche/Hitachi (1 mentions)
Lyphochek immunoassay plus control, by Bio-Rad (1 mentions)
Cytotox 96, by Promega (1 mentions)
Architect c4000, by Abbott (1 mentions)
Prism 9, by GraphPad (1 mentions)
Sulfuric acid, by Avantor (1 mentions)
Human thrombin, by Merck Group (1 mentions)
Rt pa, by Boehringer Ingelheim (1 mentions)
Sector imager 6000, by Mesoscale (1 mentions)
V plex human cytokine 30 plex kit, by Mesoscale (1 mentions)
30 protocols using sunrise reader
1
Plasma Myoglobin Quantification by ELISA
2
Evaluating Myocardial Infarction Extent
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The LAD was then reoccluded and 4% Evans blue was injected to determine the “area at risk” (AAR) as previously described [19 (link), 20 (link)]. The heart was excised under deep anesthesia, the LV was sliced at thickness of 2 mm and these slices were incubated in a 1% solution of 2,3,5-triphenyltetrazolium chloride dye for 15 min at 37°C and then fixed in 10% formalin for 20 min. AAR and infarct size were measured in a blinded manner using a planimetry method and Image J software (National Institutes of Health, Bethesda, MD, USA). AAR was expressed as a percentage of left ventricle area, and infarct size was expressed as a percentage of AAR.
Plasma concentrations of cardiac troponin I were measured using a commercially available ELISA kit (LSI Medience, Tokyo, Japan). Concentrations were measured spectrophotometrically using a standard 96-well plate reader at a wavelength of 450 nm (Sunrise™ reader; Tecan Group, Männedorf, Switzerland).
Plasma concentrations of cardiac troponin I were measured using a commercially available ELISA kit (LSI Medience, Tokyo, Japan). Concentrations were measured spectrophotometrically using a standard 96-well plate reader at a wavelength of 450 nm (Sunrise™ reader; Tecan Group, Männedorf, Switzerland).
3
Quantifying Superoxide Dismutase Activity
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MnSOD activity was detected using Superoxide Dismutase Assay kit (Cayman Chemical) following the manufacturer’s protocol. The method uses tetrazolium salt to quantify superoxide radicals generated by xanthine oxidase and hypoxanthine. The standard curve was generated by using a quality-controlled SOD standard. The absorbance was monitored at 440–460 nm using a TECAN Sunrise Reader (Tecan, Männedorf, Switzerland). One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. Enzymatic activity was presented as units/mL per milligram of total protein. The results were defined after triplicate analysis.
4
Superoxide Dismutase Enzyme Assay
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Superoxide Dismutase Assay kit (Cayman Chemical) was used to visualize MnSOD activity, following standard protocols. Evaluation of superoxide radicals, generated by xanthine oxidase and hypoxanthine, was performed with tetrazolium salt. Standard curve was generated with a quality-controlled SOD standard.
TECAN Sunrise Reader (Tecan, Männedorf, Switzerland) was used to measure absorbance at 440–460 nm. Enzymatic activity was presented as units/mL. One unit of SOD is the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. The results were defined after triplicate analysis.
TECAN Sunrise Reader (Tecan, Männedorf, Switzerland) was used to measure absorbance at 440–460 nm. Enzymatic activity was presented as units/mL. One unit of SOD is the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. The results were defined after triplicate analysis.
5
Adiponectin, Leptin, and Visfatin Levels
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Elbow venous blood was taken from the patients after 12 h of overnight fasting. Fasting adiponectin levels were measured by an enzyme‐linked immunosorbent assay (ELISA) kit (catalogue number ab222508). The ELISA plate reader (Tecan Sunrise Reader, 96‐well Microplate Reader) was applied to read the absorbance at 450 nm wavelength. Commercial radioimmunoassay kits (catalog number ab179884)were used to measure plasma leptin levels, with a standard curve range of 0–50 ng/mL. A visfatin enzyme immunoassay kit (catalogue number ab264623) was used to measure visfatin, with a standard curve range of 0.1–1000 ng/mL. Serum levels of TNF‐⍺ and IL‐6 were assessed using the ELISA kit (Jiangsu Meimian Industrial Co., Ltd, Jiangsu, China). All measurements were conducted in Shanghai Enzyme Linked Biotechnology Co., LTD.
6
Nanoparticle Cytotoxicity in Cancer Cells
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The viability of two cancer cell lines was assessed by the MTT assay. The Caco-2 and HCT-116 were seeded in 96-well plates in 200 μL of complete RPMI-1640 medium at a density of 5.0 × 103 and 3.0 × 103 cells per well, respectively, and incubated for 24 h. After exposure for 72 h with increasing doses of E171 and TiO2 60 nm (0.001; 0.01; 0.1; 1; 10; 100; 1000 mg/L), cells were incubated at 37 °C in 5% CO2. After removing the medium, 100 μL of MTT working solution (5 mg/mL in RPMI) was added to each well for 4 h at 37 °C in a humidified incubator. Then 100 μL of the solution of 2-propanol and hydrochloric acid (50 mL + 167 μL) was supplied to each well to solubilize formazan blue crystals. A cell-free system was used to exclude NPs interference with the test reagents, in which only the NPs were incubated with the test reagents, and their absorption was monitored.
The absorbance was measured at 590 nm with the ELISA Tecan Sunrise Reader according to the manufacturer’s instructions. Cell viability was calculated as follows:
The absorbance was measured at 590 nm with the ELISA Tecan Sunrise Reader according to the manufacturer’s instructions. Cell viability was calculated as follows:
7
Phenoloxidase Activity Assay in Galleria
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PO activity was measured according to previously published studies [61 (link),62 ]. Haemolymph of G. mellonella (5 μl) was collected directly to 95 μl of PHL (475 μg in PBS for all experiments except dose-dependence assay where different dilutions of PHL were used), PLL (475 μg in PBS), BSA (475 μg in PBS) or PBS (pH 7.4) and incubated for 10 min at room temperature. After incubation, 40 μl of reaction mixture was transported to the microplate well and 160 μl of 3,4-dihydroxy-dl -phenylalanine (3 mg/ml in PBS) was added as substrate. The reaction was allowed to proceed for 30 min, and absorbance was measured in 2-min intervals at 492 nm with a Sunrise reader (Tecan, Switzerland). PO activity was expressed as linear increase in absorbance per minute. For the inhibition assay, PHL was pre-incubated with 0.2M l -Fuc or Me-α-l -Fuc for 10 min at room temperature prior to mixing with haemolymph.
8
Quantification of Per a 2 Allergen in Extracts
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First, inhibition plates were prepared by blocking with 3% non-fat milk/PBS overnight at room temperature. After washing with PBST, three 2-fold dilutions of whole-body and fecal extracts (100 µL/well) and eight 2-fold dilutions of recombinant Per a 2 proteins were made in duplicate wells. Rabbit anti-rPer a 2 antibodies were added to each sample well (100 µL/well). The plates were incubated for 2 hours at 37℃.
On the other hand, 96-well assay plates were prepared by coating with 0.1 µg/well of rPer a 2 in a carbonate buffer for 2 hours at 37℃. Then, the non-reacted sites were blocked for 1 hour with 3% non-fat milk/PBS. After 2-hour incubation of the inhibition plates to allow antibody reaction with the tested extracts, the preabsorbed Per a 2 antibodies were transferred to 96-well assay plates and incubated for 2 hours at room temperature. After washing, a 1:5,000 dilution of peroxidase-labeled goat anti-rabbit IgG was added and incubated for 1 hour at room temperature, and a colored reaction was developed by the addition of ABTS (55 µg/mL in 0.1 M sodium citrate buffer, pH 4.2, containing 0.03% H2O2). The results were read at 415 nm on a Sunrise Reader (TECAN). The concentration of Per a 2 in the tested extracts were determined by comparing the reference standards and expressed in mg/g of extractable protein.
On the other hand, 96-well assay plates were prepared by coating with 0.1 µg/well of rPer a 2 in a carbonate buffer for 2 hours at 37℃. Then, the non-reacted sites were blocked for 1 hour with 3% non-fat milk/PBS. After 2-hour incubation of the inhibition plates to allow antibody reaction with the tested extracts, the preabsorbed Per a 2 antibodies were transferred to 96-well assay plates and incubated for 2 hours at room temperature. After washing, a 1:5,000 dilution of peroxidase-labeled goat anti-rabbit IgG was added and incubated for 1 hour at room temperature, and a colored reaction was developed by the addition of ABTS (55 µg/mL in 0.1 M sodium citrate buffer, pH 4.2, containing 0.03% H2O2). The results were read at 415 nm on a Sunrise Reader (TECAN). The concentration of Per a 2 in the tested extracts were determined by comparing the reference standards and expressed in mg/g of extractable protein.
9
Quantifying Intratumoral Cytokines and Chemokines
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To determine intratumoral cytokines and chemokines, tumors were harvested 16 h after the start of treatment, snap frozen in LN2, and stored for future use at −80°C. Tumor lysates were prepared using lysis buffer (Biorad) including factor 1, factor 2, and PMSF using Peqlab Cryolys tissue homogenizer in Precellys ceramic kit tubes with 1.4-mm beads. Protein levels were adjusted after quantification using a bicinchoninic acid protein assay kit (Thermo Fisher) to 10 mg/ml. Cytokines and chemokines were determined using a multiplex BioPlex Pro kit (mouse chemokine panel 33-plex and cytokine 23-plex; Biorad) or by ELISA for mouse IL-27 (Invitrogen). Readout was performed using BioPlex 200 (Biorad) or Synergy 2 (BioTek) instruments, respectively.
PK analysis of the αCSF-1R antibody clone 2G2 was performed by an in-house–made ELISA. In brief, biotinylated murine CSF-1R (Sino Biologicals) was coated onto streptavidin-coated plates (Roche) and incubated with mouse sera or purified 2G2 clone for standardization. Bound 2G2 antibody was detected by a peroxidase-conjugated goat anti-mIgG, specific for Fcγ subclass 1 (Jackson), and ABTS (Roche) using a Sunrise reader (Tecan).
PK analysis of the αCSF-1R antibody clone 2G2 was performed by an in-house–made ELISA. In brief, biotinylated murine CSF-1R (Sino Biologicals) was coated onto streptavidin-coated plates (Roche) and incubated with mouse sera or purified 2G2 clone for standardization. Bound 2G2 antibody was detected by a peroxidase-conjugated goat anti-mIgG, specific for Fcγ subclass 1 (Jackson), and ABTS (Roche) using a Sunrise reader (Tecan).
10
Cell Growth Rate Quantification by MTT
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The cell growth rate was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) at 24-h intervals. After 1 × 104 cells were seeded into 96-well plates, 0.5 mg/mL MTT was added over consecutive days for violet pellet formation by living cells. The pellets were solubilized in 200 μL of dimethyl sulfoxide. The optical density of each sample was measured at 570 nm using a microplate reader (Sunrise Reader, Tecan, Männedorf, Switzerland). Growth rate was measured as a percentage of control growth. Cells from passage 15 were used to determine population doubling time, and all experiments were repeated twice in triplicate.
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