The fluorescence nuclease activity assays were performed in reaction volumes of 10 μL, by mixing 8 μL of sample (24 h culture supernatants or control medium) with 1 μL of CaCl2 100 mM and 1 μL of F-TTprobe (50 pmol·μL−1). The reactions were incubated at 37 °C, for 30 min. Then, the reactions were stopped by adding 295 mL of PBS-/- supplemented with 10 mM EDTA. Finally, 95 mL of each sample was loaded in triplicate into 96-well black plates (96F non-treated black microwell plate, Thermo Scientific). Fluorescence intensity was measured with a fluorescence microplate reader, Synergy Neo2 Hybrid Multi-Mode Reader from Agilent BioTek (Winooski, VT, USA) using the filter settings for FAM (excitation/emission 485/528 nm). Three independent experiments were performed with each sample. The results are expressed as the mean ± SD (n = 3) of fluorescence intensity in arbitrary units (a.u.).
Synergy neo2 hybrid multi mode reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy Neo2 Hybrid Multi-Mode Reader is a compact and flexible instrument designed for a wide range of detection methods. It features a combination of detection modes, including absorbance, fluorescence, and luminescence, to support a variety of assays and applications.
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Lab products found in correlation
L3224, by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (4 mentions)
Hi fbs, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
24 well plate, by Corning (2 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (2 mentions)
Leica dm2500, by Leica Microsystems (2 mentions)
96f non treated black microwell plate, by Thermo Fisher Scientific (1 mentions)
Atp assay kit, by Promega (1 mentions)
White f96 microwell plates, by Thermo Fisher Scientific (1 mentions)
Ros glo h2o2 assay, by Promega (1 mentions)
Magmax microbiome ultra nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
Kingfisher flex system, by Thermo Fisher Scientific (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Ethidium homodimer 1, by Thermo Fisher Scientific (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Cgp37157, by Bio-Techne (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Calcium green 5n, by Thermo Fisher Scientific (1 mentions)
31 protocols using synergy neo2 hybrid multi mode reader
1
Nuclease Activity Assay Protocol
2
CRISPRi-Mediated Growth Rate Analysis
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We cultured E. coli strains harboring a non-targeting CRISPRi control overnight in 4 mL LB + 35 μg/mL kanamycin at 37°C with shaking. The next day, we washed these cultures twice into M9+Kan as described previously. We diluted cultures 1:200 into each of the 22 media conditions described in Continuous Culture and adapted cells for 4 hours at 37°C with shaking. Following adaptation, we washed cells into 4 mL of their respective supplemented media + 50 ng/mL anhydrotetracycline at OD600 = 0.005. Three 200 μL replicates of each culture were grown in a Synergy Neo2 Hybrid Multi Mode Reader (Agilent, #BTNEO2) overnight at 37°C while taking regular OD600 measurements. Growth rates were fit across replicates using a log-linear fit of exponential phase growth and averaged across triplicate measurements.
3
Measurement of IFN-β Induction by RIG-I Mutants
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The IFN-β induction assay was implemented to test RIG-I mutants as described previously (Ren et al., 2019 (link)). In brief, 500 µl of HEK293T cells at a concentration of 100,000 cells/ml in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher) supplemented with 10% heat-inactivated Fetal Bovine Serum (HI-FBS, ThermoFisher) was seeded into each well of 24-well plate (Corning). 24 hours after the seeding, the cells of each well were transfected with 3 ng of pUNO1-RIG-I, 6 ng of pRL-TK and 150 ng of IFN-β/Firefly using the lipofectamine 2000 transfection reagent (ThermoFisher). The RIG-I expression was allowed to proceed for 24 hours, at which point the cells of each well were challenged with 1 µg of RNAs using lipo2000 reagent. 12–16 hours after the stimulation, the HEK293T cells were lysed and the IFN-β induction was measured using the Dual-Luciferase Reporter Assay System (Promega) and a Synergy Neo2 Hybrid Multi-Mode Reader (Biotek). The IFN-β induction level, the relative luminescence unit (RLU, Fluc/Rluc), is the firefly luciferase activity normalized to the renilla luciferase activity. The data were further processed with GraphPad Prism.
4
Extracellular ATP and HMGB1 Release after IR
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5 × 105 cells following IR were incubated for 24 h, then extracellular ATP and HMGB1 release were measured. Extracellular ATP after IR was measured by an ATP Assay Kit (Promega, Madison, USA) based on luciferin–luciferase conversion following the manufacturer’s instructions. The chemoluminescent signal was read by a Synergy Neo2 Hybrid Multi-Mode Reader (BioTek, USA). HMGB1 in supernatant was determined by a HMGB1 ELISA kit (Biorbyt, UK) according to manufacturer’s instructions. The microplates were read using a multiskan spectrophotometer model 1510 (Thermo Fisher Scientific, Finland) for protein concentration assessment.
5
Measurement of IFN-β Induction by RIG-I Mutants
6
Viability and Proliferation of Bioprinted hDPSCs
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Live and dead staining (L3224; Thermo Fisher Scientific, Waltham, MA, USA) was performed in order to measure the viability of the bioprinted hDPSCs. The samples were stained with assay solution (0.2% v/v calcein AM and 0.05% v/v ethidium homodimer-1 in phosphate-buffered saline (PBS)) at room temperature for 45 min and then imaged using a fluorescent microscope (Leica DM2500; Leica Microsystems AG, Wetzlar, Germany). The live and dead cells were manually counted and the cell viability was calculated by dividing the number of live cells by the total cell count.
Proliferation was evaluated using alamarBlue™ Cell Viability Reagent (ThermoFisher Scientific). The samples cultured for 1, 3, and 5 days were incubated in 10% v/v alamar blue dye diluted by growth medium at 37°C and 5% CO2 for 3 h. After sampling the assay solutions in 100-μL aliquots, their fluorescence intensities (excitation: 544 nm/emission: 599 nm) were measured with a microplate reader (Synergy NEO2 Hybrid Multi-Mode Reader; Bio-Tek, Winooski, VT, USA). The measured data were normalized relative to the data collected on day 1.
Proliferation was evaluated using alamarBlue™ Cell Viability Reagent (ThermoFisher Scientific). The samples cultured for 1, 3, and 5 days were incubated in 10% v/v alamar blue dye diluted by growth medium at 37°C and 5% CO2 for 3 h. After sampling the assay solutions in 100-μL aliquots, their fluorescence intensities (excitation: 544 nm/emission: 599 nm) were measured with a microplate reader (Synergy NEO2 Hybrid Multi-Mode Reader; Bio-Tek, Winooski, VT, USA). The measured data were normalized relative to the data collected on day 1.
7
SARS-CoV-2 Pseudovirus Generation and Titration
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SARS-CoV-2 wild type and variant pseudoviruses were generated as described previously, with minor modifications. Briefly, HEK293T cells were grown to 70-80% confluency before co-transfection with the pCMV14 expression vector encoding either SARS-CoV-2 wild type or variant S gene, and a luciferase reporter plasmid (pNL4-3-R-E-luciferase, gifted by Dr. Wanbo Tai) at a ratio of 1:1 in Opti-MEM medium using polyethylenimine. After 5 h, the cell supernatant was replaced with fresh DMEM medium, and the cells were cultured for an additional 48 h at 37 °C in an atmosphere comprising 5% CO2. Pseudoviruses secreted into the supernatant were collected by centrifugation at 1000 × g for 10 min, filtered through a 0.45 µm pore-size membrane, and stored at −80 °C. To determine the SARS-CoV-2 pseudovirus titer, viral stocks were serially diluted2-fold with DMEM and added to 1.5 × 104 hACE2-293T cells per well in 96 well tissue culture plates. After incubation for 48 h at 37 °C in an atmosphere comprising 5% CO2, cell supernatants were removed, 1× lysis buffer containing luciferase substrate (75 µL/well) was added to the plates, and shaken at 40 rpm for 10 min at room temperature. Cell lysates were transferred into luminometer plates (Corning, Cat# 3917). Relative luciferase activity was measured using a Synergy Neo2 Hybrid Multi-Mode Reader (BioTek, USA)
8
H2O2 Quantification using ROS-Glo Assay
9
β-Arrestin 2 Recruitment Assay
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β-Arrestin
2 recruitment was determined by measuring BRET by using the NanoBRET
system.63 (link) BRET was measured between a full
length human M1 receptor N-terminally carrying a FLAG-tag and C-terminally
carrying a nanoluciferase. The β-arrestin 2 was N-terminally
modified with a HALO-tag and labeled with a HALO-618 fluorescent ligand
(Promega, Mannheim, Germany). Therefore, 1 × 106 HEK293T
cells were seeded in a 6 cm dish and after 20 h transiently transfected
with 1 μg of the receptor, 2 μg of β-arrestin 2,
and 1 μg of human GRK2 with the Effectene transfection reagent
in accordance to the user manual. Twenty hours after transfection,
cells were transferred from 6-well plates to 96-well plates. Cells
were counted, and 20,000 cells per well were seeded into white 96-well
plates (Brand GmbH & Co. KG, Wertheim, Germany). The next day,
BRET was measured using the Synergy Neo2 Hybrid Multi-Mode Reader
(BioTek Instruments GmbH, Bad Friedrichshall, Germany), and the BRET
ratio was corrected against buffer conditions. The highest ligand
concentration tested was 100 μM due to limited solubility in
the buffer used for BRET assays.
2 recruitment was determined by measuring BRET by using the NanoBRET
system.63 (link) BRET was measured between a full
length human M1 receptor N-terminally carrying a FLAG-tag and C-terminally
carrying a nanoluciferase. The β-arrestin 2 was N-terminally
modified with a HALO-tag and labeled with a HALO-618 fluorescent ligand
(Promega, Mannheim, Germany). Therefore, 1 × 106 HEK293T
cells were seeded in a 6 cm dish and after 20 h transiently transfected
with 1 μg of the receptor, 2 μg of β-arrestin 2,
and 1 μg of human GRK2 with the Effectene transfection reagent
in accordance to the user manual. Twenty hours after transfection,
cells were transferred from 6-well plates to 96-well plates. Cells
were counted, and 20,000 cells per well were seeded into white 96-well
plates (Brand GmbH & Co. KG, Wertheim, Germany). The next day,
BRET was measured using the Synergy Neo2 Hybrid Multi-Mode Reader
(BioTek Instruments GmbH, Bad Friedrichshall, Germany), and the BRET
ratio was corrected against buffer conditions. The highest ligand
concentration tested was 100 μM due to limited solubility in
the buffer used for BRET assays.
10
RNA Extraction from Wastewater Samples
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The wastewater samples were processed and RNA extracted according to the recommended protocol in Palmer et al. (2021) . Briefly, triplicate 50-mL aliquots for each wastewater sample were placed in a room temperature water bath for 1.5 h to thaw. Aliquots were then pasteurized at 60 °C for 1.5 h. Two-phase centrifugation at 4500 xg for 5 min at 4 °C resulted in a pellet that was subsequently resuspended in the lysis buffer provided in the MagMax Microbiome Ultra Nucleic Acid Isolation kit (ThermoFisher, Waltham, Massachusetts). Homogenization was conducted in the lysing tubes of the kit via four rounds of disruption in a FastPrep-24 (MP Biomedicals; Santa Ana, California) at 4.0 m/s for 20 s and centrifugation at 13,000 xg for 15–20 s.
RNA was extracted with the MagMax Microbiome Ultra Nucleic Acid Isolation kit according to the manufacturer's instructions utilizing the KingFisher Flex system (ThermoFisher, Waltham, Massachusetts). Extracted RNA was stored at −20 °C. The nucleic acid concentration was measured spectrophotometrically with the Synergy Neo 2 Hybrid Multi-Mode Reader (BioTek; Winooski, Vermont).
RNA was extracted with the MagMax Microbiome Ultra Nucleic Acid Isolation kit according to the manufacturer's instructions utilizing the KingFisher Flex system (ThermoFisher, Waltham, Massachusetts). Extracted RNA was stored at −20 °C. The nucleic acid concentration was measured spectrophotometrically with the Synergy Neo 2 Hybrid Multi-Mode Reader (BioTek; Winooski, Vermont).
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