A fluorescence-based KDalert GAPDH assay (Invitrogen, Life Technologies) was used to measure enzymatic activity of GAPDH protein of extracellular vesicles. The assay measures the conversion of NAD+ to NADH in the presence of phosphate and glyceraldehyde-3-phosphate (G-3-P). Under the recommended assay conditions, the rate of NADH production is proportional to the amount of GAPDH enzyme present. A fixed number of EVs were added to the substrate in a 96-well plate. A fluorescent microplate reader (Clariostar Plus, BMG Labtech) was used to acquire data using the kinetic mode setting, with λex = 560 nm and λem = 590 nm. Data were analyzed using MARS Data Analysis software (Clariostar Plus, BMG Labtech).
Clariostar plus
Manufactured by BMG Labtech
Sourced in Germany, United States, United Kingdom, Spain, Australia
The CLARIOstar Plus is a high-performance multimode microplate reader developed by BMG LABTECH. It is designed to provide accurate and reliable measurements for a wide range of assays, including absorbance, fluorescence, luminescence, and time-resolved fluorescence. The CLARIOstar Plus is capable of precise temperature control and fast read times, allowing for efficient and high-throughput data acquisition.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
96 well plate, by Corning (6 mentions)
Prism 9, by GraphPad (5 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (4 mentions)
Fluotrac microplates, by Greiner (4 mentions)
Streptavidin hrp, by R&D Systems (4 mentions)
Prism 8, by GraphPad (4 mentions)
Cellstar, by Greiner (3 mentions)
Pmirglo dual luciferse mirna target expression vector, by Promega (3 mentions)
Celltiter glo 3d cell viability assay, by Promega (3 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (2 mentions)
Microamp optical adhesive film, by Thermo Fisher Scientific (2 mentions)
Echo 525, by Beckman Coulter (2 mentions)
Warmstart colorimetric lamp 2 master mix, by New England Biolabs (2 mentions)
Viewseal sealer, by Greiner (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Black fluotrac microplates, by Greiner (2 mentions)
Dextran conjugated lysosensor yellow blue dnd 160, by Thermo Fisher Scientific (2 mentions)
Maxisorp, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter 1000 assay system, by Promega (2 mentions)
354 protocols using clariostar plus
1
Fluorescence-based GAPDH Activity Assay
2
Redox Assay of Glutaredoxins using roGFP2
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Oxidation and reduction assays using roGFP2 [41 (link),42 (link),52 ] were performed in a 96-well plate in a fluorescence plate reader (CLARIOstar® Plus, BMG Labtech). For oxidized and reduced roGFP2 controls, roGFP2 was treated for 30 min with 10 mM DTT or 10 mM H2O2 before the assay start. To assess the reduction capacities of PpGRXC5, 1 μM of untreated (oxidized) roGFP2 was pipetted into a well containing 1 μM GRX (PpGRXC5, AtGRXC1), 100 μM NADPH and 1 unit S. cerevisiae GR in 100 μl of 100 mM potassium phosphate buffer pH 7.4. After measuring for 10 cycles, a final concentration of 2 mM GSH was added automatically by the injection needles of the plate reader into the respective wells. Fluorescence was followed until roGFP2 ratio stabilized. For assessing oxidation capacities of PpGRXC5, 10 μM roGFP2 was pre-reduced with 10 mM DTT for 30 min and subsequently desalted via Zeba™Spin Desalting Columns (ThermoFisher) following the manufacturer's instructions. 1 μM of pre-reduced roGFP2 was mixed with 1 μM of PpGRXC5 or AtGRXC1 in potassium phosphate buffer pH 7.4. Two mM GSSG was added via the injection needles of the plate reader after 5 min of initial measurements and the measurement continued until roGFP2 ratio stabilized. Fluorescence intensities were collected by excitation at 390-10 nm or 480-10 nm and emission at 530-10 nm.
3
Extracellular ATP measurement in BeWo cells
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For measurements of extracellular ATP, BeWo cells were cultured in 96-well dishes (Nunc) with a density of 1 × 105 cells/well overnight and afterwards transfected with CD39- and control plasmid, as described above. After 24 h of transfection, the control as well as the CD39-overexpressing cells were treated with pooled platelet releasate (as described above). RealTime-Glo™ Extracellular ATP Assay Reagent was added to the treatment in a final volume of 25% (v/v) to each well and luminescence was measured via CLARIOstarPlus (BMG Labtech) every 5 min for 5 h. CD39 overexpression was confirmed by gene expression analysis of ENTPD1 in each experiment.
4
Quantifying β-Glucans in R. globosum
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R. globosum was processed for β-glucans using a commercial β-glucan assay (Yeast & Mushroom) (K-YBGL, Megazyme) following the manufacturer's protocol. Briefly, samples were processed by acid hydrolysis then enzymatic break-down and β-glucans were quantified spectrophotometrically with a CLARIOstar Plus microplate reader (BMG Labtech), relative to a negative control and positive β-glucan standard. A sample of shop-bought baker's yeast was used as an additional positive control.
5
Phage LUZ100 Bacteriolytic Activity Quantification
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The bacteriolytic activity of phage LUZ100 was determined by monitoring the growth of the phage-infected bacteria over time. For this, overnight cultures of three biological replicates of PaLo41 were inoculated in fresh LB medium and incubated at 37°C to an OD600 of 0.3. Next, these cultures were infected with LUZ100 at an MOI of either 1 or 10. The OD600 of the uninfected and infected cultures was measured every 15 min for 145 min on the CLARIOstar Plus microplate reader (BMG Labtech, Ortenberg, Germany) for four technical replicates during incubation at 37°C. To assess the lytic activity of LUZ100, the phage score (PS) and virulence index (Vϕ) were determined (19 (link), 20 (link)). For this, the area under the infection curve (A) was determined using the statistical software JMP. The following formulas were applied, for which the virulence curve represents a plot of vMOI as a function of the MOI (NC = negative control):
6
Cytotoxicity Evaluation of Infected Precision-Cut Lung Slices
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To assess the viability of infected and mock PCLSs, 50 μL of supernatants (infected with 10³ TCID50 IDV and/or 10³ CFU M. bovis ) were collected at different time points and were tested using the Pierce LDH cytotoxicity assay kit (Thermo Scientific), following the manufacturer’s instructions. Positive controls for LDH release were created by incubating PCLSs with a lysis buffer provided by the kit for 1 h at 37°C (“maximum LDH activity”). The optical density of positive controls and PCLS supernatants was read at 490 and 680 nm (background) using a CLARIOStar Plus plate reader (BMG LabTech). The 680-nm absorbance value was subtracted from the 490-nm absorbance before calculating the percentage of cytotoxicity, using the formula [(LDH at 490 nm) − (LDH at 680 nm)] for each sample (“PCLS sample LDH activity”). The percentage of cytotoxicity was then calculated using the following formula: [(PCLS sample LDH activity)/(maximum LDH activity)] × 100.
7
Cell Viability Assay with PrestoBlue
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Cells were seeded in flat-bottom 24-well plates (Greiner, Frickenhausen, Germany) 24 h prior to treatments. Five days after treatment, cells were incubated with DMEM containing 10% PrestoBlue™ Cell Viability Reagent (Invitrogen, Waltham, MA, USA) for four hours, and fluorescence was measured with a CLARIOstar® Plus microplate reader (BMG Labtech, Ortenberg, Germany) using excitation/emission wavelengths of 560 ± 15/590 ± 20 nm. Blank measurements were included for background correction.
8
Evaluating bEnd.3 Cell Barrier Tightness
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The paracellular permeability of fluorescein isothiocyanate (FITC) labeled 4 kDa dextran (FD4, Sigma Aldrich) to determine the tightness of our bEnd.3 model.
The bEnd.3 cells were cultured with rat astrocytes as described above. On the day of the experiment, the inserts were transferred to a new astrocyte-free plate, and the medium was changed on both the apical and basolateral sides. To initiate the permeability experiment and avoid the temporary disruption of the barrier, 50 μL medium was removed from the apical side and 50 μL 200 μM working solution of 4kDa FITC-dextran was added to the upper well, i.e. the apical well had 500 μl 10 μM 4kDa FITC-dextran. In every 15 min for a period of 60 min, starting from 0 min, 100 μl medium was removed from the basolateral side. At the final time point, 100 μl medium was also removed from the apical compartment. The fluorescence intensity of the sample was read by a CLARIOstar® Plus microplate reader (BMG Labtech) (λExc = 485 nm, λEm = 515 nm, Bandwidth = 15 and 20 nm, respectively), and the paracellular permeability was calculated according to Eqs (1 ) and (2 ). The permeability experiments were repeated three times in technical triplicates, and the data are represented as mean (SD).
The bEnd.3 cells were cultured with rat astrocytes as described above. On the day of the experiment, the inserts were transferred to a new astrocyte-free plate, and the medium was changed on both the apical and basolateral sides. To initiate the permeability experiment and avoid the temporary disruption of the barrier, 50 μL medium was removed from the apical side and 50 μL 200 μM working solution of 4kDa FITC-dextran was added to the upper well, i.e. the apical well had 500 μl 10 μM 4kDa FITC-dextran. In every 15 min for a period of 60 min, starting from 0 min, 100 μl medium was removed from the basolateral side. At the final time point, 100 μl medium was also removed from the apical compartment. The fluorescence intensity of the sample was read by a CLARIOstar® Plus microplate reader (BMG Labtech) (λExc = 485 nm, λEm = 515 nm, Bandwidth = 15 and 20 nm, respectively), and the paracellular permeability was calculated according to Eqs (
9
Quantifying Protein-DNA Interactions
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SpRY and Cas9 gRNP complexes were prepared as described above, and serial 2-fold dilutions were incubated with 0.8 nM FAM-labeled scrambled DNA for 3 h at 37 °C in 1 x Cas9 buffer supplemented with 0.05% Tween-20. Data were recorded at 37 °C in a CLARIOstar Plus multi-detection plate reader (BMG Labtech) equipped with a fluorescence polarization optical module (λex = 485 nm; λem = 520 nm). The data were fit using a one-step binding model in KinTek Explorer to define the Kd and the extrapolated starting (Y0) and ending (ΔY) points, which were used to normalize the data for calculation of fraction bound (Fig. 3c ). The value of ΔY from the SpRY data was also used to normalize the Cas9 data: and
10
Intestinal Permeability Measurement Using TRITC-Dextran
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At indicated, intestinal permeability was assessed by oral gavage of mice with 500 mg/kg body weight TRITC-dextran (4 kDa) dissolved in saline containing 0.5% carboxymethylcellulose. Blood was collected at the tail tip 4 h after gavage and plasma was used to determine TRITC fluorescence intensity. Standard curves were used to evaluate TRITC (excitation: 544 ± 10 nm; emission: 580 ± 10 nm) concentrations and fluorescent intensity in plasma of each sample was measured using the microplate reader CLARIOstar® Plus (BMG Labtech, Ortenberg, Germany).
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