The same mouse from each group was imagined at the appointed time by using an IVIS imaging system. Briefly, infected mice were anesthetized with isoflurane and 100 μl Nano-Glo reagent (Promega, dilution 1:25) was injected into retro-orbitally of mice. The Living Image software was used for image acquisition and analysis. Flux measurements were automatically calculated from the signal of mice. All data of composite images used the same scale.
Nanoglo reagent
Manufactured by Promega
Sourced in United States
NanoGlo is a luminescent reagent used for in vitro detection and quantification of reporter gene activity. The reagent contains an engineered luciferase that generates a bright, stable luminescent signal upon interaction with a substrate.
Automatically generated - may contain errors
Lab products found in correlation
Ivis spectrum ct in vivo imaging system, by PerkinElmer (2 mentions)
Living image software, by PerkinElmer (2 mentions)
Mouse anti flag antibody, by Merck Group (2 mentions)
Glo lysis buffer, by Promega (2 mentions)
Flexi rabbit reticulocyte lysate system, by Promega (2 mentions)
Glomax 96 plate reader, by Promega (2 mentions)
Nanoluc luciferase, by Promega (1 mentions)
Anti flag hrp, by Merck Group (1 mentions)
Expifectamine 293 reagent, by Thermo Fisher Scientific (1 mentions)
Centro xs3 lb 960 luminometer, by Berthold Technologies (1 mentions)
Tristar2 lb 942 multimode plate reader, by Berthold Technologies (1 mentions)
384 well plate, by Greiner (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Bugbuster, by Merck Group (1 mentions)
Nanoluc luciferase reporter gene, by Promega (1 mentions)
Spectramax, by Molecular Devices (1 mentions)
Rfp trap ma, by Proteintech (1 mentions)
Gfp trap ma, by Proteintech (1 mentions)
Halt protease inhibitor mix, by Thermo Fisher Scientific (1 mentions)
Benzonase, by Merck Group (1 mentions)
19 protocols using nanoglo reagent
1
Bioluminescent Imaging of Infected Mice
2
Quantification of NanLuc Luciferase Activity
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The Nano-Glo® reagent (Promega, Madison, WI, USA), which produces ATP-independent luminescence upon the oxidation of furimazine by the NanLuc luciferase, was used to measure presence of NanLuc luciferase according to the instructions from the manufacturer. An aliquot of 20 μL of Nano-Glo® substrate was added to 1 mL of Nano-Glo® buffer. The resulting reagent was vortexed and either used immediately or stored at 4 °C for later use.
3
Quantifying Protein-Protein Interactions via LUMIER
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LUminescence-based Mammalian IntERactome (LUMIER) assay was carried out by transfecting HEK293 cells in 96-well plates with plasmids that expressed each client protein fused to NanoLuc luciferase (Promega) along with 3XFLAG-tagged HSP90α and HSP90β. After 18 hours, cells were washed with cold PBS and lysed with 100 μl TGNET complete buffer, then centrifuged at maximum speed for 15 minutes at 4°C. The resulting lysates were transferred to an anti-FLAG antibody-coated plate (Sigma) and incubated at 4°C for 2 hours with gentle shaking. The plates were washed with TGNET. Nano-Glo reagent (Promega) was added and luciferase activity was quantified using a plate reader (Perkin-Elmer). To normalize for 3XFLAG-HSP90 levels, the plates were washed again with TGNET and anti-FLAG-HRP (Sigma-Aldrich) was added. The plates were once again incubated at 4°C, washed and assayed for HRP luminescence activity. The experimental relative interaction strength for each client protein interaction was determined by dividing the NanoLuc readout by the HRP readout value. Each sample was assayed 3 times with 3 replicates. Standard deviations are represented by error bars. A two-tailed T-test was used to determine statistical significance. All calculations were performed in Excel (Microsoft).
4
RBD-ACE2 Binding Inhibition Assay
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3.2 ng RBD-Smbit (residues 319–541, C-terminal tag) was diluted in 20 µL 0.02% BSA/PBS and mixed with 20 µL mAb dilution (17 µg/mL) or mAb fragment dilution (corresponding molarity) in 96-wells. After 1 h incubation at RT, ACE2-LgBit was added in volume of 20 µL so that 10 × molar excess over the RBD was achieved. After 1 h incubation at RT, 60 µL NanoGlo reagent (Promega) was added and luminescence quantified as with the mix-and-read assay. Wells where antibody dilution was substituted with the diluent served to determine the baseline signal of RBD-ACE2 binding.
5
Quantifying cdiG and cGAMP Signaling
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G. sulfurreducens strains with either the cdiG- or the cGAMP-Nanoluc reporter integrated into the Tn7 site were grown to mid-log fumarate-limited medium (40 mM acetate and 80 mM fumarate), the same ratio of acetate:fumarate as in RNA-seq conditions, and lysed at room temperature for 5 min in a phosphate-buffered saline (PBS) solution containing 1 × BugBuster (Novagen) and 0.3 mg/ml DNase. 10 μl of the Nanoglo reagent (Promega) and 10 μl of the cell lysate were combined in a white-bottom, 96 well plate and luminescence was detected at 461 nm (Molecular Devices). Biological replicates (n = 3) were assayed. In assays where the luminescence exceeded the linear range of the spectrophotometer or deviated from steady-state, lysates were diluted in PBS before combining with the Nanoglo reagent.
6
Transfection and Luciferase Assay in HEK-293F
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HEK-293F cells were grown in suspension and transfected with ExpiFectamine 293 reagent (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s recommendations. Briefly, a total of 75.106 cells were harvested and the pellet was resuspended in 25 mL of Expi293 Expression medium before incubation at 37 °C for 30 min. Twenty-five micrograms of each plasmid were diluted in 1.5 mL of Opti-MEM medium and mixed with ExpiFectamine reagent before incubation at room temperature for 20 min. Finally, the mixed ExpiFectamine/plasmid was added to the cells and further incubated at 37 °C under agitation for 4 days. ExpiFectamine Transfection enhancers were added on day 1 post-transfection. Recombinant proteins were harvested directly from the culture supernatant at day 4 post-transfection. Luciferase activity was quantified onto a Centro XS3 LB 960 luminometer (Berthold Technologies, Thoiry, France) by adding 100 µL of NanoGlo reagent (Promega, Madison, WI, USA) to tenfold dilutions of supernatant.
7
Nano Luciferase Reporter Assay for RE Signaling
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Stably expressing response element (i.e. NFκB-RE, CRE, AP1, SRE, SRF-RE, SIE) driven Nano luciferase SW1353 (HTB-94, ATCC) reporter cell lines generated according to previously published protocol (Ripmeester et al., 2020 (link)). Reporter cells were Trypsinized (Trypsin; ThermoFisher) and re-seeded (60.000 cells/cm2) into 384-well plates (Greiner Bio-One) and cultured overnight in DMEM/F12 supplemented with 0.5% FCS. Serum-starved cells were stimulated with 10% extracellular vesicles for 6 h. After stimulation, cells were lysed using 15 µL Milli-Q. Following the addition of Nano-Glo reagent (1:1 ratio; Promega), luminescence was quantified using the Tristar2 LB942 multi-mode plate reader (Berthold Technologies).
8
Bioluminescent Imaging of VEEV Infection
9
Quantification of Cellular Luciferase Activity
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Dense TYG pre-cultures were created as described above for growth curves. TYG cultures were diluted 100-fold into prewarmed MM with 0.5% carbohydrate solution in borosilicate glass tubes. After 24 h incubation at 37 °C under anaerobic conditions, cells were pelleted by centrifugation and the supernatant was discarded. Cells were lysed by resuspending in 1/10th culture volume of BugBuster® (Millipore Sigma) and incubating at room temperature for 10 min. Phosphate buffered saline (PBS) was used to dilute lysate 20 to 200-fold to ensure the signal was within the dynamic range of the luminescence detector. 30 μL samples were added to an opaque 96 well microplate and combined with 30 μL of freshly prepared NanoGlo reagent (Promega). Luminescence was measured using a Synergy HT Multi-detection plate reader. Technical replicates, each being the average of three observations, were tested for each condition. The luminescence of the negative controls (30 μL PBS + 30 μL NanoGlo) was used to normal signal for each replicate. The detected value corrected for the dilution factor and normalized to the density of the original culture as shown in Eq. (1 ).
The technical triplicates were then averaged and plotted with the standard error of the mean.
The technical triplicates were then averaged and plotted with the standard error of the mean.
10
SARS-CoV-2 Luciferase Assay Protocol
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Luciferase assays were performed using a molecular complementary DNA clone of a SARS-CoV-2 isolate (USA/WA-1/2020) in which a nonessential gene (ORF7) was replaced by the NanoLuc luciferase reporter gene (Promega), as previously described for SARS-CoV-1 and MERS-CoV (74 (link)). Virus infectivity assays were conducted in 96-well tissue culture plates. Vero E6 cells were seeded at a density of 2 × 104 cells per well in DMEM supplemented with 10% FBS (DMEM/FBS) and incubated for 15 to 24 hours. The next day, serial dilutions of antibodies or human IgG1 isotype control were prepared in DMEM/FBS. The SARS-CoV-2–NanoLuc inoculum was diluted in DMEM/FBS, mixed with an equal volume of diluted antibody to produce a final virus titer of 140 plaque-forming units (PFU) per well, and incubated for 1 hour. After removing the culture medium from the plated Vero E6 cells, the virus-antibody solution was inoculated onto duplicate wells and incubated for 48 hours. Following standard protocols as recommended by the vendor, Nano-Glo reagent (Promega, #N1110) was added and luciferase activity was quantified on a SpectraMax plate reader (Molecular Devices).
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