To express influenza virus HA fused to GLuc or NLuc, HEK293FT cells (ATCC, Manassas, VA) maintained in Dulbecco’s modified Eagle medium (Gibco) containing 10% fatal bovine serum (FBS) were transfected with purified plasmid DNA using Polyethylenimine MAX (Polysciences, Inc., Warrington, PA); the transfection was performed using 70 to 90% confluent cells grown in six-well plates. Twenty-four hours after transfection, cells were rinsed with phosphate-buffered saline without CaCl2 and MgCl2 [PBS(−)], and chilled Renilla lysis buffer (Promega) was distributed at 200 μl/well. Cells then were harvested with a cell scraper, and the lysate from each well was transferred to a 1.5-ml microfuge tube and centrifuged at 13,000 × g for 10 min at 4°C. The resulting supernatant was transferred to a fresh tube and stored at −80°C until use.
Renilla lysis buffer
Manufactured by Promega
Sourced in United States
Renilla Lysis Buffer is a solution used for the lysis of cells expressing the Renilla luciferase reporter gene. It is designed to efficiently release the Renilla luciferase enzyme from the cells, allowing for its measurement and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Tristar lb 941 luminometer, by Berthold Technologies (5 mentions)
Renilla luciferase assay system, by Promega (5 mentions)
Renilla luciferase kit, by Promega (5 mentions)
Fluostar omega plate reader, by BMG Labtech (5 mentions)
Luciferase substrate reagent, by Promega (4 mentions)
Renilla luciferase reagent, by Promega (2 mentions)
Renilla luciferase substrate, by Promega (2 mentions)
Centro lb960 luminometer, by Promega (2 mentions)
Hek293ft cells, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine max, by Polysciences (1 mentions)
Coelenterazine, by Promega (1 mentions)
Centro lb960, by Promega (1 mentions)
Enspire plate reader, by PerkinElmer (1 mentions)
Renilla luciferase assay kit, by Promega (1 mentions)
Centro lb 960, by Berthold Technologies (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Centroxs luminometer, by Berthold Technologies (1 mentions)
Streptactin beads, by GE Healthcare (1 mentions)
Renilla luciferase assay reagent, by Promega (1 mentions)
36 protocols using renilla lysis buffer
1
Expressing Influenza HA with Luciferase Tags
2
RSV Antiviral Assay in A549 Cells
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A549 (CCL-185, ATCC) cells were seeded at 1.6 × 104 cells in 100 μL of 5% FBS/1X penicillin–streptomycin/F12 medium per well in 96-well µClear® black plates with a clear bottom (Greiner 655090). The cells were infected with 400 PFU/well of a recombinant RSV encoding Renilla luciferase as an additional transcription unit (rA2-Rluc [22 (link)]) in a total volume of 50 µL/well and allowed to adsorb for 1 h. Purified compounds were serially diluted two-fold in triplicate and 50 µL/well of the diluted extracts were added to the infected cells. After further incubation at 37 °C and 5% CO2 for 24 h, the supernatants were removed and cells were lysed using Renilla lysis buffer (Promega). Luciferase activity was measured with the Renilla Luciferase reagent (Promega) and BioTek Synergy Mx microplate luminometer using Gen5 version 2.00.18 software. The RSV antiviral effect for the test samples was determined by normalizing to the DMSO-treated control samples and multiplying by 100 to obtain the percent of control. Statistical analysis was performed using GraphPad Prism software version 9.4.1.
3
LUMIER Binding Assay of Protein Interactions
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LUMIER binding assays with pairs of putative interactors, one fused to luciferase and the other fused to GST, were performed in LUMIER lysis buffer (150 mM NaCl, 0.1% Triton X-100, 20 mM Tris-HCl (pH 7.4), 5% glycerol, 5 mM EDTA and proteinase inhibitors) as previously described (Ryzhakov and Randow, 2007 (link)). GST-fusion proteins were immobilized on beads before incubation with the luciferase tagged binding partner for 2 h. Luciferase-tagged proteins were expressed in 293ET cells. After washing in lysis buffer, proteins were eluted with glutathione in Renilla lysis buffer (Promega). Relative luciferase activity represents the ratio of activity eluted from beads and present in lysates.
4
HCoV-229E-Luc Infection Assay in Huh-7 Cells
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HCoV-229E-Luc was first mixed with the compounds at the appropriate concentrations. On the day before infection, 6 × 104 Huh-7 cells and Huh-7/TMPRSS2 cells were seeded in 96-well plates at 37 °C. The cells were inoculated with HCoV-229E-Luc at a MOI of 0.5 in a final volume of 50 μL for 1 h at 37 °C in the presence of the different compounds, either at 25 μg/mL for the screening experiment or at increasing concentrations for the dose–response experiment. The virus was removed and replaced with culture medium containing the different compounds for 6 h at 37 °C. The cells were lysed in 20 μL of Renilla Lysis Buffer (Promega, Madison, WI, USA), and luciferase activity was quantified using a Tristar LB 941 luminometer (Berthold Technologies, Bad Wildbad, Germany) with the Renilla Luciferase Assay System (Promega) as recommended by the manufacturer.
5
Gaussia Luciferase Complementation Assay
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For the GPCA (Gaussia Protein Complementation Assay), 2.5 × 104 HEK293T cells were seeded in 96-well plates. After 24 h, cells were transfected with 100 ng of pSPICA-N2 (encoding G2 fragment of Gaussia princeps luciferase), pSPICA-N2-HPV16-L2, or pSPICA-N2-HPV5 L2 or pSPICA-N2-BPV1-L2 and 100 ng of pSPICA-N1 (encoding G1 fragment of Gaussia princeps luciferase) or pSPICA-N1-PLK1wt, pSPICA-N1-PLK1 T210D, pSPICA-N1-PML, pSPICA-N1-HPV16 L1, pSPICA-N1-IRF3, pSPICA-N1-E6AP. At 24 h post transfection, cells were washed with PBS and lysed with Renilla Lysis Buffer (Promega). Gaussia princeps luciferase enzymatic activity was measured using a Berthold Centro LB960 luminometer by injecting coelenterazine (Promega) and counting luminescence for 10 s. Results were expressed as normalized relative luminescence (RLUC). For a given proteins pair A/B, RLUC = (G1−A + G2−B)/[(G1−A + G2) + (G1 + G2−B)]70 (link).
6
Quantifying HCV and DENV Infection
7
ZIKV Infection Assay with NITD008
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5 × 104 HeLa cells were plated in 24-well plates. Next day, cells were transfected with control siRNA or FMR1_2 siRNA. After 46 hr of knockdown, cells were treated with 0.05% DMSO or 20 µM NITD008 for 2 hr before infection with the ZIKV reporter at an MOI of 3. NITD008 or DMSO were retained in media during the infection. After 1 hr of incubation, inoculum was retired and replaced with fresh media containing DMSO or NITD008. 2.5 hr later, cells were washed five times with 1 × PBS and lysed with Renilla lysis buffer (Promega). Additionally, cycloheximide (CHX) treatment (200 µM) was used as a background control in absence or presence of NITD008. For this condition, cells were pretreated with CHX (in absence or presence of NITD008) for 2 hr before ZIKV reporter infection and CHX was retained during infection until cell lysis. Luciferase assays were performed using the High-Affinity NanoBit evaluation system (Promega) and the Enspire plate reader (Perkin Elmer).
8
Split-Luciferase Assay for PB1-ANP32A Interaction
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Split-luciferase assays were undertaken in 293Ts seeded in 24-well plates. We cotransfected 30 ng each of PB2, PA, and PB1, with the N terminus of Gaussia luciferase (Gluc1) tagged to its C terminus after a GGSGG linker cotransfected using Lipofectamine 3000 along with ANP32A, tagged with the C terminus of Gaussia luciferase (Gluc2) on its C terminus (after a GGSGG linker). Twenty-four hours later, cells were lysed in 100 μl of Renilla lysis buffer (Promega), and Gaussia activity was measured using a Renilla luciferase kit (Promega) on a FLUOstar Omega plate reader. Normalized luminescence ratios (NLRs) were calculated by dividing the values of the tagged PB1 and ANP32 wells by the sum of the control wells, which contained (i) untagged PB1 and free Gluc1, and (ii) untagged ANP32A and free Gluc2 as described elsewhere (15 (link), 37 (link)).
9
Antiviral Activity Screening of Juncus acutus Against HCoV-229E
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Hepatoma cell line Huh-7 and Huh-7 cells transduced with a lentivirus encoding for TMPRSS2 protease, a cellular protease that allows fusion of the virus at the host cell surface (Huh-7/TMPRSS2), were used for all HCoV-229E infection assays. All the above-mentioned samples from Juncus acutus stems were screened for their antiviral activity against HCoV-229E-Luc expressing the luciferase, a recombinant HCoV-229E with luciferase reporter gene. Huh-7 cells seeded in 96-well plates were inoculated with HCoV-229E-Luc in the presence of each extract, fraction or compound for 1 h. Inoculum was then removed and replaced with culture medium containing the compounds for another 6 h. Finally, cells were lysed in 20 µL of Renilla lysis buffer (Promega) and luciferase activity was quantified using Renilla luciferase assay kit (Promega). Luciferase activity was measured by the use of a Tristar LB941 luminometer (Berthold Technologies, Bad Wildbad, Germany).
10
Luminescence-based Transfection Assay
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HEK-293T cells were seeded into white 96-well plates at 3 × 104/well. After 24 h, cells were transfected with linear PEI (polyethylenimine) with 300 ng of a Glc2-PB2-expressing plasmid and 100 ng of a Glc1-UPS-expressing plasmid. At 24 h posttransfection, cells were washed with 100 µl of phosphate-buffered saline and lysed with 40 µl of Renilla lysis buffer (Promega E2820) for 1 h. G. princeps luciferase enzymatic activity was measured with a Berthold Centro LB960 luminometer by injecting 50 µl of luciferase substrate reagent (Promega E2820) per well and measuring luminescence for 10 s. Results were expressed in relative luminescence units.
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