NIH3T3 reporter cells were infected with MCMV, treated with IFNβ or IFNλ for 6 h and lysed for quantification of reporter gene expression (Le‐Trilling et al, 2018 (link)). Luciferase activity was measured according to the manufacturer's instructions (pjk) using a microplate multireader (Mithras2 LB 943; Berthold Technologies). Quantification of luminescence was performed by use of the microplate multireader Mithras2 LB 943 and MicroWin software (Berthold Technologies). The resulting data were analysed using GraphPad Prism software. The values are reported as mean ± standard deviation (SD) as described in the figure legends.
Mithras2 lb 943
Manufactured by Berthold Technologies
Sourced in Germany, Switzerland
The Mithras2 LB 943 is a multimode microplate reader designed for a wide range of applications in life science research. It offers advanced detection technologies, including absorbance, fluorescence, and luminescence measurements. The device is capable of performing various assays, such as cell-based, biochemical, and ELISA experiments, among others. The Mithras2 LB 943 provides accurate and reliable data to support scientific investigations.
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Lab products found in correlation
Microwin software, by Berthold Technologies (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
G3582, by Promega (1 mentions)
Luciferase assay reagent, by Promega (1 mentions)
Nano glo hibit lytic detection system, by Promega (1 mentions)
Lb 941 luminometer, by Berthold Technologies (1 mentions)
Nanoluciferase, by Promega (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Phospha light system, by Thermo Fisher Scientific (1 mentions)
Glomax discover microplate reader, by Promega (1 mentions)
Catalase, by Merck Group (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Superoxide dismutase sod, by Merck Group (1 mentions)
Sensoplate, by Greiner (1 mentions)
Nano glo luciferase substrate, by Promega (1 mentions)
18 well μ slides, by Ibidi (1 mentions)
26 protocols using mithras2 lb 943
1
Quantification of MCMV-Induced Luciferase
2
Characterizing miRNA-target interactions
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HLA-DPB1 3′UTR fragments or control wild-type (WT) and mutant (mut) target sequence of hsa-miR-21 (mir21-WT and mir21-mut) were pre-amplified by PCR (primers and conditions in Table 3 ) or synthetized in vitro (Eurofins Genomics, Ebersberg, Germany). 3′UTR fragments and controls were cloned into the pmirGLO vector (Promega, Madison, WI, USA) downstream of the luciferase reporter gene (luc2) and transfected into HeLa cells or BLCL by electroporation with the Neon transfection system (Invitrogen, USA), according to the manufacturer's recommendations. Luciferase activity was measured after 24 h with a Dual Luciferase Reporter Assay System (Promega) using the monochromator multimode microplate reader LB 943 Mithras2 (Berthold Technologies, Bad Wildbad, Germany). Luciferase activity under the control of mir21-WT or mir21-mut was used as positive and negative controls, respectively, since the expression of the relevant miRNA hsa-miR-21 was shown to be abundant in both HeLa and BLCL (24 (link), 25 (link)). The Renilla luciferase gene (hRluc-neo fusion) included in the same vector was used as transfection control for normalization of the luc2 signal.
3
RBOH Activity Measurement and Localization
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Measurements of RBOH activity were performed as previously (Drerup et al., 2013; Fujita et al., 2020; Han et al., 2019) . Each well was transfected with 100 ng of total plasmid (50 ng pEF1-RBOH, 30 ng pEF1-PM-LKS4 and empty pEF1-vector to ensure equal transfection efficiency and loading). 0.1 mM of the phosphatase inhibitor Calyculin A was added directly before the start of the measurements. To observe Ca 2+ -dependent activation of RBOHB CaCl 2 (1 mM final concentration) and Ionomycin (1 mM final concentration) were injected10 min after starting the measurement to induce Ca 2+ influx. Luminescence was measured using a LB 943 Mithras 2 (Berthold) microplate reader and is presented as relative luminescence units S-1 (RLU S-1). For all experiments, R 3 replicates were measured in parallel and mean±SD was calculated. Three independent experiments confirmed all presented data.
For localization studies of LKS4 and PM-LKS4 in HEK293T cells, pEF1-LKS4-mCherry and pEF1-PM-LKS4-mCherry were used for transfection. Adherent cells were detached via treatment with Trypsin-EDTA solution in PBS (Sigma) for five minutes at 37 C. Trypsin digestion was stopped by adding 5x volume of HBSS and the cell suspension was used for microscopic investigation. Fluorescence was observed using the 63x objective of a Leica SP5 confocal microscope (excitation, 561 nm; emission, 585-635 nm) .
For localization studies of LKS4 and PM-LKS4 in HEK293T cells, pEF1-LKS4-mCherry and pEF1-PM-LKS4-mCherry were used for transfection. Adherent cells were detached via treatment with Trypsin-EDTA solution in PBS (Sigma) for five minutes at 37 C. Trypsin digestion was stopped by adding 5x volume of HBSS and the cell suspension was used for microscopic investigation. Fluorescence was observed using the 63x objective of a Leica SP5 confocal microscope (excitation, 561 nm; emission, 585-635 nm) .
4
BRET Assay for Monitoring PTEN Interactions
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BRET investigations were performed as described previously (Lima-Fernandes et al., 2014 (link)). Briefly, HEK cells were transfected with the indicated plasmids 24 hr after seeding. At 24 hr post transfection, cells were detached, resuspended in full media, and distributed into poly-l-orthinine coated white 96-well optiplates (Perkin Elmer). The following day, cells were washed with PBS and then overlayed with HBSS. Coelenterazine h was added to a final concentration of 5 mM and incubated for 3 min at 25°C. BRET readings were collected using a Multimode Reader Mithras2 LB 943 (Berthold Technologies). Substrate and light emissions were detected at 480 nm (Rluc) and 540 nm (YFP) for 1 s. The BRET signal was calculated by ratio of the light emitted by YFP and the light emitted by Rluc (YFP/Rluc). The ratio values were corrected by substracting background BRET signals detected when Rluc-PTEN was expressed. mBRET values were calculated by multiplying these ratios by 1000. ∆mBRET values are shown to demonstrate the shift in BRET signal compared to wt signal, which is set to zero, or between the two mutants (C124S-T383A and C124S-A4) that were tested.
5
Colorimetric MTS Viability Assay
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Metabolic activity of viable cells was quantified colorimetrically using a standard in vitro viability MTS assay according to the manufacturer's instructions (Promega, G3582) (Fig. 1 I2). The exposure liquid was removed apically and on the basolateral side the RPMI medium was replaced with 1 mL phenolred free RPMI medium (Sigma-Aldrich, R7509). To each insert, 360 µL MTS-media solution (1 part MTS stock and 5 parts phenolred free RPMI medium) was added apically. After 30–40 min at standard cultivation conditions, 3 times 100 µL supernatant (triplicates) was pipetted into 96-well plates to determine the absorbance at 490 nm using a plate reader (Berthold Technologies, Mithras2 LB943, Fig. 1 I2). Each acute toxicity assay contained negative controls (NC, n = 3) by which only RPMI medium (70 µL) was added to the A549 cell monolayer apically and absorbance readings were set to 100% viability for comparison of the other absorbance values (positive control and textile extracts, SI Table S8). The sensitivity of the assay was assessed with positive controls containing cadmium sulfate (CS) at three different concentrations each in triplicates (1 mM; 10 mM; 50 mM) in RPMI medium. The acute toxicity assay was repeated 3 times with different A549 cell passages (P13-P15, biological replicates, n = 3) and 3 independent washing extracts (N = 3), see SI Table S8.
6
Quantifying TGF-β1 in Cell Cultures
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The concentration of TGF-β1 in the cell culture medium was determined using an ELISA kit (R&D Systems; cat. no. DB100B). Cell culture medium supernatant was assayed following the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Mithras2 LB943, Berthold Technologies), and chemokine concentrations were quantified using MikroWin 2010 software (version 5.14, Labsis Laborsysteme GmbH).
7
Quantification of Luciferase Reporter Assays
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Firefly luciferase was analyzed by adding 50 μl Luciferase Assay Reagent (Promega) to 5 μl of translation mixture after cell-free protein synthesis. The concentration of active firefly luciferase was determined using a calibration curve. Luminescence was detected using the LB 941 luminometer (Berthold Technologies). Nanoluciferase (Promega) activity was detected using the Nano-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. Briefly, 100 μl of reagent was added to 100 μl of cells and incubated for 5 min at 300 rpm. For cell-free produced Nanoluciferase 5 μl translation mixture after cell-free protein synthesis was mixed with 50 μl reagent and incubated for 3 min. Detection of HiBiT (Promega)-tagged eIF2α-S52A in CHO cells was achieved using the Nano-Glo HiBiT Lytic Detection System (Promega) according to the manufacturer's instructions. Briefly, 100 μl Nano-Glo HiBiT Lytic Reagent containing buffer, substrate and LgBiT for luciferase complex formation was added to 100 μl cell suspension and incubation was performed for 10 min at 300 rpm. The luminescence signal of the HiBiT and Nanoluciferase assay was detected by the Multimode Microplate Reader Mithras 2 LB 943 (Berthold Technologies) using an OD2 filter.
8
Neutralization of Adenovirus Serotypes
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In brief, HEK293 cells were seeded into 96-well plates at 2 × 104 cells per well. Two days later, serial dilutions of macaque sera or mAbs were incubated with HAdV55-SEAP, HAdV11-SEAP, or HAdV14-SEAP at 2 × 106 v.p. per well at 37°C for 1 h. The mixtures were added to the plates and incubated at 37°C for 24 h. Finally, the culture supernatants were harvested and SEAP activities were measured using the Phospha-Light System (Thermo Fisher Scientific). Relative light units (RLU) were recorded by GloMax Discover Microplate Reader (Promega). The neutralizing titres of immune sera were calculated as the reciprocal of the dilutions at which 50% of RLU values were inhibited. The IC50 values of mAbs were calculated similarly.
mAb targeting sites were analysed by neutralization assay based on HAdV55-EGFP, HAdV11-EGFP, HAdV14-EGFP, as well as chimeric HAdV14-EGFP. Each mAb was diluted to 100 ng/ml and incubated with each virus (2 × 106 v.p. per well) at 37°C for 1 h. The mixtures were added to A549 cells in 96-well plates. After incubation at 37°C for 24 h, cells were fixed in 10% neutral buffered formalin (NBF) for 10 min. EGFP signals were excited at 488 nm and detected at 509 nm by Multimode Reader Mithras2 LB 943 (Berthold) and recorded as RLU. The neutralizing activities of the mAbs against each HAdV were calculated as Neutralization % = (RLUvirus-RLUmAb.)/ RLUvirus × 100%.
mAb targeting sites were analysed by neutralization assay based on HAdV55-EGFP, HAdV11-EGFP, HAdV14-EGFP, as well as chimeric HAdV14-EGFP. Each mAb was diluted to 100 ng/ml and incubated with each virus (2 × 106 v.p. per well) at 37°C for 1 h. The mixtures were added to A549 cells in 96-well plates. After incubation at 37°C for 24 h, cells were fixed in 10% neutral buffered formalin (NBF) for 10 min. EGFP signals were excited at 488 nm and detected at 509 nm by Multimode Reader Mithras2 LB 943 (Berthold) and recorded as RLU. The neutralizing activities of the mAbs against each HAdV were calculated as Neutralization % = (RLUvirus-RLUmAb.)/ RLUvirus × 100%.
9
Extracellular TPO Activity in Thyrocytes
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Considering that TPO is expressed at the surface of the cell, we determined its extracellular enzymatic activity as previously described [15 (link)]. Human primary thyrocytes were seeded in 6-well plates and treated without or with palmitic acid (0.2 or 0.4 mM) for 72 hours. The cells were washed twice with 2 ml of PBS, then 0.5 ml of reaction mixture was added (100 μM KI (Sigma-Aldrich), 200 U/ml superoxide dismutase (SOD, Sigma-Aldrich), and 50 μM Amplex Red (Life Technologies) in sodium phosphate buffer). The reaction was initiated by adding 25 μl of 1 mM H2O2 (Sigma-Aldrich). 20 μl of aliquots were removed at 1-minute intervals during 8 minutes and immediately mixed with 80 μl of inhibition mixture containing 500 U/ml catalase (Sigma-Aldrich) and 100 U/ml SOD in PBS. The fluorescence was measured in a microtiter reader (Mithras2 LB 943, Berthold, Bad Wildbad, Germany) using excitation at 530 nm and emission at 590 nm. Enzymatic activity values were normalized with corresponding protein amount.
10
MCMV and HCMV Mutants Expression
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Previously described MCMV and HCMV mutants expressing enhanced green fluorescent protein (eGFP) were employed [23 (link)]. BJ-5ta, MLN4924-resistant BJ-5ta (BJ-5ta-RMLN), CIM, and MRC-5 cells were seeded in 96-well microplates with black rim for fluorescence determinations. Cells were infected with indicated virus doses (0.1 and 1 PFU/cell), applying centrifugal enhancement (900 g, twice for 15 min). Fluorescence was visualized using a BioSys Bioreader 7000-F-z-i after 24, 48, and 72 h and/or quantified using a microplate multireader (Mithras2 LB 943; Berthold Technologies, Bad Wildbad, Germany).
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