Cell-cell fusion assays were performed using the double split protein system, as previously described (50 (link)), with some modifications. 293T cells were seeded in six-well plates at 300,000 cells per well and transfected with Mirus TransIT-293. Cells regarded as producer cells, expressing ecotropic glycoprotein with or without IFITM3, were transfected with pCD-Env (1.2 μg), pCMV-IFITM3 (0.84 μg or 3-fold dilutions thereof), and plasmid expressing DSP1-7 (0.2 μg). Cells regarded as targets were transfected with plasmid expressing DSP8-11 (0.2 μg) and pcDNA-mCAT1 (2.4 μg). At about 24 h posttransfection, producer cells were detached, and 50 μl of the cell suspension was transferred to a 96-well plate in triplicate. At 48 h posttransfection, target cells were incubated with 60 μM Enduren (Promega) and detached, and 50 μl of the cell suspension was mixed with producer cells, resulting in a 30 μM final concentration of Enduren. The luciferase activity was measured 2 h after mixing producer and target cells.
Enduren
Manufactured by Promega
Sourced in United States, Switzerland, Germany
EnduRen is a bioluminescent reporter assay reagent designed to measure long-term gene expression in live cells. It utilizes a luciferase enzyme that is engineered for enhanced stability and extended luminescent signal, allowing continuous monitoring of reporter gene activity over extended time periods.
Automatically generated - may contain errors
Lab products found in correlation
Multidrop dispenser, by Thermo Fisher Scientific (5 mentions)
Centro xs960 luminometer, by Berthold Technologies (4 mentions)
Nutridoma sp, by Roche (4 mentions)
Trypsin, by Corning (4 mentions)
Cellstripper, by Corning (4 mentions)
Spectramax i3x microplate reader, by Molecular Devices (4 mentions)
Infinite f200 reader, by Tecan (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Celltiter glo, by Promega (2 mentions)
Prism 5, by GraphPad (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Glomax luminometer, by Promega (1 mentions)
Transit 293, by Mirus Bio (1 mentions)
M200 pro microplate reader, by Tecan (1 mentions)
Transit mrna transfection reagent, by Mirus Bio (1 mentions)
Lv200, by Olympus (1 mentions)
M1000 pro, by Tecan (1 mentions)
F200 pro, by Tecan (1 mentions)
M1000 pro plate reader, by Tecan (1 mentions)
Pe ivis spectrum, by PerkinElmer (1 mentions)
34 protocols using enduren
1
Cell-Cell Fusion Assay with IFITM3
2
SARS-CoV-2 Entry Inhibition Assay
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For the DSP assay using 293FT cells, effector cells expressing S protein with DSP8-11 and target cells expressing CD26 or ACE2, and TMPRSS2 with DSP1-7 were seeded in 12-well cell culture plates (2 × 105 cells/500 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen (Promega, Madison, WI, USA), a substrate for Renilla luciferase, to activate EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in dimethyl sulfoxide (DMSO) was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 μL of each single cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Scientific, Waltham, MA, USA). After incubation at 37 °C for 4 h, the RL activity was measured using a Centro xS960 luminometer (Berthold, Germany). For the DSP assay using Calu-3 or H3255 cells, target cells were seeded in 384-well plates (2 × 104 cells/50 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in DMSO was added to the 384-well plates with 9 μL of culture medium. Next, 40 μL of single cell suspension (effector cells) was added to the wells using a Multidrop dispenser.
3
MERS-CoV Membrane Fusion Quantification
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MERS-CoV-S-mediated membrane fusion was quantitatively evaluated via DSP assay using 293FT cells as previously described (Yamamoto et al., 2016 (link)). The dimerization of DSP1-7 and DSP8-11 after cell-cell fusion can be quantified based on the values of fluorescence/luminescence upon formation of tight DSP complexes. The effector cells express MERS-CoV-S protein with DSP8-11, while the target cells express the MERS-CoV receptor and transmembrane serine protease 2 (TMPRSS2) with DSP1-7. The cells were grown in 10 cm culture dishes (4 × 106 cells/10 ml) 24 h before the assays. Cells were treated with 6 µM EnduRen (Promega, Madison, WI, United States), a substrate for Renilla luciferase, for 2 h to activate EnduRen. Each peptide was dissolved in dimethyl sulfoxide (DMSO) and added to 384-well plates (Greiner Bioscience, Frickenhausen, Germany), then 50 µl of each single-cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Fisher Scientific). After incubation at 37°C for 4 h, luciferase activity was measured using a Centro xS960 luminometer (Berthold, Germany).
4
Quantitative Cell-Cell Fusion Assay
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The cell-cell fusion assay was performed as described previously (Yamamoto et al., 2016 (link)). To quantitate the cell-cell fusion, a pair of 293FT-based reporter cells, effector and target cells, that express individual split reporters (DSP1-7 and DSP8-11 proteins) were used, because DSP1-7 and DSP8-11 produce fluorescence and luminescence only when the two proteins form a tight complex (Wang et al., 2014 (link)). The effector cells stably expressing DSP8-11 and S-protein and the target cells stably expressing DSP1-7 together with CD26 and TMPRSS2 were prepared. 2 h before the fusion assay, both cells were treated with 6 μM EnduRen (Promega, Madison, WI, USA), a substrate for Renilla luciferase, to activate EnduRen. 1 mL of each compound dissolved in dimethyl sulfoxide (DMSO) was added to 384-well plates (Greiner Bioscience, Frickenhausen, Germany) using a 12-stage workstation (Biotech, Tokyo, Japan). Next, a Multidrop dispenser (Thermo Scientific, Waltham, MA, USA) was used to add 50 μL of each single cell suspension (1.5×104 effector and target cells) to the wells. Incubation was performed at 37°C for 4 h, then RL activity measurements were obtained with a microplate reader (PHERAStar Plus, BMG Labtech, Cary, NC, USA).
5
SARS-CoV-2 Membrane Fusion Assay
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The DSP assay using 293FT cells was performed as described previously (Yamamoto et al., 2020 (link)) to monitor SARS-CoV-2-S-mediated membrane fusion. Briefly, effector cells expressing SARS-CoV-2-S protein with DSP8-11, target cells expressing ACE2, and transmembrane serine protease 2 (TMPRSS2) with DSP1-7 were seeded in 10 cm culture dishes (4×106 cells/10 mL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 µM EnduRen (Promega, Madison, WI, USA), a substrate for Renilla luciferase, to activate EnduRen. One microliter of each peptide dissolved in dimethyl sulfoxide (DMSO) was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 µL of each single-cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Fisher Scientific, Waltham, MA, USA). After incubation at 37°C for 4 h, luciferase activity was measured using a Centro xS960 luminometer (Berthold, Bad-Wildbad, Germany).
6
Quantifying SARS-CoV-2 S Protein Fusion
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The fusion activity of the S protein with ACE2 was measured as previously described. In brief, 293T cells were co-transfected with S protein (25 ng) and a dual-split protein 1-7 (DSP1-7) expression (25 ng) vectors using Lipofectamine 3000 in a 96-well plate. The 293T/ACE2 cells were simultaneously transfected with DSP8-11 (2.5 μg) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) in a 6-well plate. Twenty-four hours following transfection, 293T/ACE2 transfected with DSP8-11 were treated with EnduRen (Promega, Madison, WI, USA) at a concentration of 6 μM for 2 h. Subsequently, the cells were co-cultured with 293T cells expressing S and DSP1-7 at a 2:1 ratio for 3 h. The Renilla luciferase activity was quantified using a GloMax luminometer (Promega, Madison, WI, USA). The fusion activity of the S protein was quantified by calculating the luciferase activity relative to that of the wild-type S protein, which was set at 100%.
7
Quantifying Hsp90-Cdc37 Interaction
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Complementation‐based bioluminescence assay (SRL‐PFAC) was used to explore the interaction between K292Q Hsp90 and S13D Cdc37. The fragment (1‐229) of Renilla luciferase (RL) from pGL5 vector (Promega, Madison, WI, USA) was fused to the N terminus of Hsp90 harboring the K292Q mutation and the fragment (230‐343) of RL was fused to the C terminus of Cdc37 harboring S13D mutation. MDA‐MB‐231 cells expressing those plasmids were lysed followed by treatment with different compounds for 1 hour, and the substrate EnduRen (Promega) of RL was added to the reaction mixture. Luciferase activity was determined by M200Pro microplate reader (Tecan, Seestrasse, Männedorf, Switzerland).
8
Replicon Assays for Viral Replication
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Replicon assays were performed essentially as described in [120 (link)]. Briefly, purified replicon RNA was transfected into HeLa cells grown on 96 well plates using TransIT mRNA transfection reagent (Mirus), and the cells were incubated in the growth medium supplemented with 5μM of cell-permeable Renilla luciferase substrate EnduRen (Promega) at 37°C directly in an ID3 multiwell plate reader (Molecular Devices). The measurements were taken every hour for 18 hours, the data were processed using GraphPad Prism software. Translation assay with a replicon RNA with the Δ3D mutation was perfomed similarly, but with 5x more RNA used for transfection. Total replication or translation signals were calculated as the area under the curve for the kinetic luciferase measurement for each well, the signal from at least 16-wells was averaged for each sample, unpaired t-test was used to compare the differences within pairs of experimental and control conditions, p<0.05 was considered significant.
9
Single-cell Bioluminescence Circadian Monitoring
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Real-time bioluminescence in the Nluc-WT and Nluc-TKO cells was monitored at the single cell level using an LV200 (Olympus, Japan) as described previously62 (link). EnduRen (Promega) was used as the substrate of NLuc. Values were normalized to maximum peak intensity of sample of WT cells kept in the darkness over time and to average intensity over time. To test the significance of the circadian rhythmicity and acrophase, we performed computerized analysis of normalized data in “Cosinor” and “Acro” software downloaded from the Circadian Rhythm Laboratory Software home page (http://www.circadian.org/softwar.html ).
10
Receptor Internalization Monitoring by BRET
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Plasmids encoding receptors or β-arrestins fused to RLuc were cotransfected with either KRas-Venus, Rab5-Venus, Rab7-Venus, or Rab11-Venus. Then, 24 h post-transfection, cells were collected and seeded in 96-well microplates (165306, Nunc) and cultured for an additional 24 h. Cells were then incubated for at least two hours with 5 µM Enduren (Promega) before stimulation with 100 nM h or m chemerin. BRET1 signal between RLuc and Venus was measured at 37 °C to favor receptor internalization. BRET readings were collected using an Infinite F200 reader (Tecan). The BRET signal was calculated as the ratio of emission of Venus (520–570 nm) to RLuc (370–480 nm).
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