Infinite m200 pro reader
Manufactured by Tecan
Sourced in Switzerland, Austria, United Kingdom
The Infinite M200 Pro is a multi-mode microplate reader designed for flexible and efficient sample analysis. It features a monochromator-based optical system that allows for absorbance, fluorescence, and luminescence measurements across a wide range of wavelengths. The instrument is capable of performing various microplate-based assays in life science research and clinical applications.
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75 protocols using infinite m200 pro reader
1
Kinetics of PLK Phosphorylation Assay
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PL -probes were phosphorylated using SaPLK as described previously (Hoegl et al., 2018 (link)). UV/Vis-spectra of PL and PL -probes, as well as their phosphorylated counterparts were recorded in assay buffer (20 mM HEPES pH 7.0, 100 mM KCl, 1 mM MgCl2) at 37°C using an InfiniteM200 PRO reader (TECAN, Cat# IN-MNANO). The initial rate of product formation was measured by following the increase in absorbance at the maxima of PLP (392 nm) and PLP -probes (PL1P : 416 nm, PL2P : 396 nm, PL3P : 400 nm). A typical assay contained 0.25 μM hPLK in assay buffer and activity was monitored at 37°C using an InfiniteM200 PRO reader (TECAN, Cat# IN-MNANO) in the presence of 1 mM ATP. PL and PL -probes were added from 100x stocks in dimethylsulfoxide (DMSO) to varying concentrations including no enzyme controls. Three biological replicates with three technical replicates each were performed.
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2
Measuring Biomarkers in Blood and Saliva
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Capillary blood glucose was measured immediately using a HemoCue Glucose monitor (HemoCue AB, Ängelholm, Sweden). Blood for insulin analysis stood at room temperature for 30 min before being centrifuged for 10 min to separate the plasma portion to be stored at − 40 °C. Plasma insulin concentrations were determined using enzyme-linked immunosorbent assay (Mercodia Insulin ELISA, Mercodia, Uppsala, Sweden) in which samples were pipetted in duplicate and internal and external controls were used. Absorbance was read using a Tecan M200 Infinite Pro Reader (Tecan, Switzerland). To measure salivary α-amylase activity, fasting saliva samples were collected during the first visit with the use of a saliva collection tube (Salivette, Sarstedt AG & Co, Numbrecht, Germany), centrifuged and stored at − 20 °C. Activity was measured using an enzymatic assay (1-1902; Salimetrics, State College, PA). On the day of analysis, saliva samples were thawed, centrifuged, and diluted to a 1:200 dilution. Absorbance was measured at 405 nm at 1 and 3 min using a Tecan M200 Infinite Pro Reader (Tecan, Switzerland). Activity was calculated as the change in absorbance from 1 to 3 min. Absorbances were converted to units of activity/ml of salivary α-amylase as indicated by the kit producer.
3
Crocin Toxicity Screening in Mammalian Cells
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An alamarBlue assay was used to assess the toxicity of crocin to confluent and preconfluent mammalian cells in vitro [22 (link)]. HFFs and RH cells were seeded into 96-well cell culture plates in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2. To detect the growth-inhibiting effects on confluent cells, HFFs and RH cells were seeded at densities of 10,000 cells per well and 50,000 cells per well, respectively. After overnight culture, crocin was added and diluted in serial 1:10 serial dilutions down to 100 μM. To detect growth-inhibiting effects on proliferating cells, HFFs and RH cells were seeded at densities of 1000 and 5000 cells per well, respectively. Crocin was added after 6 h of cell attachment. After treatment for 5 days, cell viability was measured using an alamarBlue assay. The cells were washed three times in PBS, and resazurin was added to 10 mg/L. Fluorescence at 595 nm was measured at 0 and 4 h with an Infinite M200 Pro reader (Tecan). The values obtained at 0 h were subtracted from those obtained at 4 h. IC50 values were calculated using an online IC50 calculator (https://www.aatbio.com/tools/ic50-calculator ) after logit-log transformation, and averages and SDs of six independent setups were calculated.
4
Cell Viability and Proliferation Assay
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The Cell Counting Kit-8 Assay (CCK-8 kit, E606335, Sangon Biotech Co. Ltd., Shanghai, China) was used to measure cell toxicity and proliferation. To determine cell toxicity, cells were cultured in 96-well plates by 1*10⁴ before they were administered with different doses of Sal003 (0, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 h. To ascertain proliferation, the culture of cells was performed in 96-well plates at 2.5 × 10³ with previous concentrations and were administered Sal003 for 24, 48, and 72 h. Fresh complete media containing 10 μL of CCK-8 reagent was added into plates according to specific time periods, and the cells were incubated at 37°C for 2 h. Absorbance at 450 nm (optical density values, OD) was assessed using an Infinite M200 Pro reader (Tecan Life Sciences, Männedorf, Switzerland).
5
Bacterial Killing by Neutrophils under pH Variation
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A bacterial killing assay with human neutrophils and opsonized S. aureus (ATCC 25923) was performed as previously described (49 (link)). Shortly, 10 × 106 neutrophils per milliliter were incubated for 30 min in HEPES- or bicarbonate-buffered medium with different pH values. Also, 9 × 105 PMN were then co-incubated for 30 min at 37°C with 9 × 106 opsonized bacteria under different pH values in bicarbonate- or HEPES-buffered media. Following co-incubation, cells were lysed and the bacterial growth/survival was measured in a Tecan infinite M200 Pro reader (Tecan) on basis of changes in optical density (OD). Series of 1:2 dilutions from the stock bacterial suspension were measured in parallel and used to calculate the percentage of bacterial survival. The different pH values did not impair the viability and did not affect the growth kinetics of S. aureus (data not shown).
6
Quantifying Chemerin Levels in Uterine Flushing
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Chemerin concentrations in the culture media were determined using a commercial ELISA kit (FineTest, Wuhan, China) following the manufacturer’s protocol. The range of standard curve was 0.156–10 ng/mL. The sensitivity of the assay was approximately 0.1 ng/mL. The sensitivity of the assays was defined as the lowest protein concentration that could be differentiated from zero samples. Absorbance was measured at 450 nm with the use of Infinite M200 PRO reader with Tecan i-control software (Tecan, Switzerland). The data were linearized by plotting the log of chemerin concentration versus the log of the optical density, and the best fit line was determined by regression analysis. Intra- and inter-assay coefficients of variation of the assay were 3.22% ± 0.39 and 8.1%, respectively.
The basal concentrations of chemerin in the uterine luminal flushing were determined by [23 (link)] and are detailed in theSupplementary Table S1 .
The basal concentrations of chemerin in the uterine luminal flushing were determined by [23 (link)] and are detailed in the
7
TRAP-enriched RNA Extraction and Quality Assessment
8
Cytotoxicity Assay for Neurospheres
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Cytotoxicity was assessed by measuring the lactate dehydrogenase (LDH) release from cells with damaged membranes using the CytoTox-ONE Homogeneous Membrane Integrity assay (#7891, Promega, Madison, WI, USA). For this, parallel to the substance testing on the MEA, one Brainphere was placed in the middle of each well of a coated 96-well plate (for coating see Supplementary Table S1 ) and differentiated in CINDA+ for 4 weeks. After acute treatment with the respective substance (15 min at 37 °C), 50 µL medium from each well was transferred to a new 96-well plate and 50 µL CytoTox-ONE reagent was added. As lysis control, neurospheres were treated with 10% Triton-X 100. The medium without spheres was used to correct for background fluorescence. Fluorescence was detected with the Tecan infinite M200 Pro reader (ex: 540 nm; em: 590 nm).
9
Measurement of Hepatic TrxR1 Activity
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The TrxR1 enzyme was measured in the extracts from the liver of male white outbred rats homogenised in 50 mM of PBS containing 1 mM of EDTA and centrifuged for 15 min at 10,000 rpm at 4 °C. The supernatant was adjusted to 7.5 µg/mL of protein as determined with pyrogallol red. Then, 40 μL of the enzyme solution was mixed with 10 μL of the test compounds in a 96-well clear flat bottom plate. After 10 min at 25 °C, 30 μL of PBS containing 0.7 mg/mL of BSA and 0.8 mM of NADPH was added. After 15 min at 25 °C, 20 µL of DTNB solution was added to the desired final concentrations. Kinetic studies were carried out by assaying TrxR1 at various concentrations of DTNB. The optical density of the samples was measured with an Infinite M200 Pro reader (Tecan, Grödig, Austria) at 412 nm every 30 s for 20 min. The activity of TrxR1 was calculated as a reaction slope relative to the control wells using Prism 8.0 (GraphPad, Inc., San Diego, CA, USA).
10
Measuring Oxidative Stress in hNPCs
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Accumulation of reactive oxygen species (ROS) was measured using 2′,7′-dichlorofluorescein diacetate (DCFDA; Sigma-Aldrich, #D6883). Therefore, 35 neurospheres with a defined size of 0.1 mm were plated per well of black/clear bottom PDL/laminin-coated 96-well plates (Thermo Fisher, #165,305) and treated with TM for 60 h. As a positive control, cells were incubated with 0.01 mM H2O2 for 45 min at 37 °C and 5% CO2. After treatments, hNPCs were washed with 100 µL prewarmed PBS and cultured in 100 µL differentiation medium containing 50 µM DCFDA for 30 min at 37 °C and 5% CO2. Afterwards, hNPCs were washed on ice using 100 µL refrigerated PBS and fluorescence was determined at 493em/522ex on a Tecan Infinite M200 Pro reader.
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