Viral stocks were treated with MβCD at various concentrations at 37 °C for 1 h followed by ultracentrifugation to remove the MβCD. The MβCD-treated virus supernatants were purified through a 20% sucrose cushion (wt/vol) prepared in TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA] by centrifugation at 36,900 rpm for 1 h at 4 °C in a P70AT rotor (model CP100WX; Hitachi, Hitachinaka, Japan). The virion cholesterol content was determined using fluorescence intensity analysis. Briefly, 96-well plate wells were coated with 50 ng of the purified viruses in 50 mM sodium bicarbonate buffer (pH 9.6) and incubated at 4 °C overnight. Plates were washed three times with washing buffer (0.05% Tween-20 in PBS) and blocked with 5% powdered skim milk (BD Biosciences, Belford, MA) in PBS at 37 °C for 2 h. After washing, filipin III (Cayman Chemical, Ann Arbor, MI) was added in triplicate for 1 h in the dark. The plates were washed, and fluorescence intensity was measured with a SPARK 10M multimode microplate reader (TECAN, Männedorf, Switzerland). In parallel, the purified samples were used to infect PAM-pCD163 or Vero cells for 48 h, and the virus-infected cells were independently subjected to FACS analysis and virus titration to determine PRRSV or PEDV infection as described above.
Spark 10m multimode microplate reader
Manufactured by Tecan
Sourced in Switzerland, United States
The Spark 10M is a multimode microplate reader designed for versatile laboratory applications. It features a monochromator-based optical system that allows for flexible wavelength selection and precise absorbance, fluorescence, and luminescence measurements.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Miseq platform, by Illumina (3 mentions)
Tcs sp8 x confocal microscope, by Leica (2 mentions)
Calcein am, by BD (2 mentions)
Calf thymus dna, by Merck Group (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (2 mentions)
Poly l lysin, by Merck Group (2 mentions)
96 well opaque plate, by Thermo Fisher Scientific (2 mentions)
Picoprobe acetyl coa fluorometric assay kit, by Abcam (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Ex taq dna polymerase, by Takara Bio (2 mentions)
Nucleospin soil kit, by Macherey-Nagel (2 mentions)
Filipin 3, by Cayman Chemical (1 mentions)
Cp100wx, by Hitachi (1 mentions)
78 protocols using spark 10m multimode microplate reader
1
Viral Cholesterol Content Quantification
2
Characterization of Fluorescent Probes
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Solvents and chemicals were purchased from commercial sources and used directly without further purification. Fast‐pfu DNA polymerase, Seamless Cloning and assembly kit, and restriction enzymes were purchased from TransGen (Beijing, China). 1H NMR spectra were recorded on a Varian 400 MHz NMR spectrometer. 13C NMR spectra were recorded on a Varian 400 MHz NMR spectrometer. ESI‐HRMS data were measured on Thermo LCQ Deca XP Max mass spectrometer. Silica gel flash column chromatography was performed on Biotage Isolera one. Fluorescence emission spectra and full wavelength absorption spectra were recorded on Tecan Spark™ 10 m Multimode Microplate Reader. Confocal fluorescence images were acquired with Leica TCS SP8 X Confocal Microscope.
3
ELISA Assay for e1 Peptide Antibodies
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After coating 96-well NuncMaxiSorp™ plates overnight at 4°C with e1 peptide diluted at 10 µg/ml in 25 mM borate buffer (pH 9), wells were blocked with PBS Tween-20 0.05% (PBST) and 5% BSA for 2 h at 37°C, incubated with mouse (1:500–1:2000) or human (1:2000) sera in PBST and 0.1% BSA for 2 h at 37°C. After three PBST washes, the corresponding secondary antibody coupled to HRP (1:2000; Jackson ImmunoResearch) was incubated for 1 h at 37°C. TMB substrate (Sigma Aldrich) was added, and the reaction was stopped by adding 50 µl H2SO4 0.5 M. Optical density was read at 450 nm using Spark10M multimode microplate reader (Tecan). For human samples, sera were processed in duplicate with 2–3 plates per series. In each plate, eight serial dilutions of a pooled serum internal control from e1-immunized mice and of a human serum internal control (pooled from three multiple sclerosis samples) served as positive day-to-day and plate-to-plate controls. Human specimens were considered high responders (HR) when the optical density exceeded the cutoff value, set at 2.5 SD above the HV mean.
4
Split Cre Reporter Assay in HEK293 Cells
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Plasmids encoding EF1α-driven fluorescent target proteins and N- and C-terminal split Cre chimeric variants were transfected by calcium phosphate into HEK293 cells (AAV293 cells purchased from Agilent Technologies, Inc., Santa Clara, CA, USA) along with plasmids encoding pAAV-CAG-FLEX-NanoLuc. Between 100 and 200 ng of total DNA was transfected into single wells of 96-well plates. Cells were ~80–100% confluent at the time of transfection. Cells were harvested 1 day (Figs. 1 and 3 ) or 2 days (Fig. 3f ) later for the Nano-Glo Luciferase Assay System (Promega). All transfections were done at equal plasmid molar ratios. We used a Spark10M multimode microplate reader (TECAN, Männedorf, Switzerland) to detect luminescence. For the experiments for which results are shown in Supplementary Fig. 2 , an mCherry-expressing HEK293 cell line was purchased from Applied StemCell (AST-1320). This stable cell line was generated via the integration of mCherry into the TARGATT HEK293 Master cell line with the use of a unique integrase and an mCherry control plasmid.
5
ROS Monitoring Using H2DCFDA Assay
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For ROS monitoring, the H2DCFDA assay (ThermoFisher Scientific, Waltham, MA, USA) was used. Nonfluorescent H2DCFDA was converted by oxidation into fluorescent 2′,7′-dichlorofluorescein (DCF), which is used as an indicator of ROS production. THP-1 cells were seeded in a black 96-well plate with a flat bottom, 3 × 104 cells per well, in a medium containing PMA and differentiated as described above. After the recovery period, the cells were washed and incubated with LPS, and resuspended SiO2NFs in the medium at concentrations of 0, 10, 100, and 200 µg/mL for 6 or 24 h at 37 °C, 5% CO2. The supernatant was then discarded, and the cells were washed. The reagent was diluted in PBS to a final concentration of 10 µM and added to each well. After 15 min of incubation, the fluorescence intensity was measured with a Spark™ 10 M multimode microplate reader (Tecan, Männedorf, Switzerland).
6
Endothelial Cell Migration Assay
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HUVECs (5 × 104 per well) were seeded into black 96-well plates with a clear bottom (Corning Costar, Amsterdam, the Netherlands) and grown overnight to a confluent monolayer. After stimulation of endothelial cells with TNF-α (50 ng/mL) for 24 h, RMS cells were incubated with 2.5 μM calcein AM (BD Pharmingen) for 30 min at 37°C in cell culture medium, washed, allowed to rest for 30 min, and treated with 100 ng/mL SDF-1 for 15 min or 20 ng/mL HGF for 30 min, or they were in control medium without growth factors. 1 × 104 RH30 cells were added to the endothelial monolayers and incubated at 37°C for 15 min. Plates were washed three times with PBS to remove unbound cells, and fluorescence was read with a fluorescence plate reader (Spark 10M multimode microplate reader, Tecan) at an excitation wavelength of 495 nm and an emission wavelength of 515 nm. The results were normalized to the percentage of the cells under control conditions.
7
Characterization of Cy-KUE-OA Nanoparticles
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Kolliphor RH40 was purchased from Baden aniline and soda factory. Compound S2 was purchased from Xi'an confluore Biological Technology Co., Ltd. All other chemicals were purchased from Beijing Innochem Science & Technology Co., Ltd., and commercially available reagents were used directly. Prostate Specific Membrane Antigen (D7I8E) XP® Rabbit mAb #12815 were obtained from Cell Signaling Technology (Danvers, MA, USA). Cy-KUE-OA particles were characterized by Dynamic Light Scattering (DLS) from Malvern Instruments Ltd. Fluorescence absorption spectra and emission spectra were determined on Tecan Spark™ 10M Multimode Microplate Reader. Confocal laser scanning microscope imaging was conducted using a Leica TCS SP8 X Confocal Microscope. All 1H NMR spectra were recorded at 400, 500 or 600 MHz, and 13C NMR spectra were recorded at 100, 125 or 150 MHz (JEOL, JEOL ECZ-400S; Zhongke-Niujin, WNMR-I 500; Bruker, AVANCE III HD 600), respectively. Mass spectra (MS) were recorded on a Thermo LCQ Deca XP Max mass spectrometer using electrospray ionization (ESI) modes.
8
Endothelial Cell Migration Assay
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HUVEC endothelial cells (5 × 104 per well) were seeded in black 96-well plates with a clear bottom (Corning Costar, Amsterdam, The Netherlands) and grown overnight to a confluent monolayer. After stimulation of endothelial cells with TNF-α (50 ng/mL) for 24 h, RMS cells were incubated with 2.5 μM Calcein AM (BD Pharminogen, Franklin Lakes, NJ, USA) for 30 min at 37 °C in cell culture medium, washed, then they rested for 30 min and treated with 100 ng/mL SDF-1 for 15 min or 20 ng/mL HGF for 30 min or they were in control medium without factors. 1 × 104 RMS cells were added to the endothelial monolayers and incubated at 37 °C for 15 min. Plates were washed three times with phosphate–buffered saline to remove unbound cells and the fluorescence was read using a fluorescence plate reader (Spark™ 10M multimode microplate reader, Tecan, Männedorf, Switzerland) with excitation at 495 nm and emission intensity detected at 515 nm. The results were normalized to percentage of the cells in control conditions.
9
Evaluating radiosensitivity of cancer cells
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Hormone depletion was carried out by washing the cells with phenol red-free IMEM (GIBCO Cat# A10488-01) without serum (five washings daily, 1 hour apart). Cells were cultured in phenol red-free IMEM supplemented with 5% charcoal-dextran-treated calf serum (Valley Biomedical Cat# BS3050) and 2% penicillin-streptomycin in a T150 flask. Cells were trypsinized and 1 × 104 cells were seeded in 6-well tissue culture plates. 24 hours after treatment with vehicle or OTX015 (1 μM), cells were subjected to IR treatment (Mark 1 137Cs irradiator, J.L. Shepherd and Associates) at various doses as indicated in each figure. Cells were then allowed to grow in an incubator for 7 days. On day 7, cells were washed, fixed with 4% paraformaldehyde, stained with 0.5% crystal violet solution for 20 minutes, washed with water, and air dried. After photographic images of the plates were obtained, crystal violet was solubilized with 20% acetic acid and absorbance was quantified by spectrophotometry at 590 nm using a Spark 10 M multimode microplate reader (Tecan).
10
Dual-Luciferase Assay for YAP/TAZ Activity
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iPSC-derived neurons (2 × 104 cells) were transfected with 10 μg of 8xGTIIC-luciferase reporter (34615, Addgene, Watertown, MA, USA) and 10 μg of pGL4.74[hRluc/TK] Vector (E6921, Promega, Madison, WI, USA) using Lipofectamine LTX with Plus Reagent (14338100, Thermo Fisher Scientific, Waltham, MA, USA). The 8xGTIIC-luciferase reporter plasmid possesses eight synthetic TEAD-binding sites upstream of the luciferase gene, making it YAP/TAZ-responsive; this construct was generated by adding four more TEAD-binding sites to 4XGTIIC-Lux, originally created by Ian Farrance58 (link) (https://www.addgene.org/34615/ ). pGL4.74[hRluc/TK] encodes the luciferase reporter gene hRluc (Renilla reniformis). After 48 h transfection, an equal volume of Dual-Glo Luciferase Reagent (E2920, Promega, Madison, WI, USA) was added to each well. Firefly luminescence was measured on a Spark 10 M multimode microplate reader (TECAN, Männedorf, Switzerland) after incubation for 20 min. For Renilla luminescence, an equal volume of Dual-Glo Stop & Glo Reagent (E2920, Promega, Madison, WI, USA) was added to each well before Dual-Glo Luciferase Reagent, mixed, and measured on a Spark 10 M. For the recovery experiments, AAV-CMV-YAPdeltaC-ins61 or AAV-CMV-NINS (MOI: 5000) or 20 nM S1P (S9666, Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium 4 days before plasmid transfection.
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