rMP12delNSs:hRen growth curves were performed in Aag2 cells. For this, 1.7 × 105 Aag2 cells per well were seeded in 24-well plates and left to adhere overnight. Cells were infected at a multiplicity of infection (MOI) of 1, and virus growth was monitored at the indicated time points by plaque assay, luciferase activity assay, and qRT-PCR. Plaque assays were performed using cell supernatant as described above. Infected cell monolayers were lysed using passive lysis buffer (Promega). Luciferase assays were performed using Renilla-Glo substrate (Promega) on a GloMax luminometer. For qRT-PCR, cells were lysed in TRIzol reagent (Life Technologies, Inc.). qRT-PCRs were performed to determine rMP12delNSs:hRen hRen and nucleocapsid (N) mRNA copy numbers in infected Aag2 cells using random hexamer primers and a QuantiTect Probe PCR kit according to the protocol of the manufacturer (Qiagen) with the following primers and probe: for hRen FP, hRen RP and hRen probe (hRen); for RVF FP, RVF RP and RVF probe (RVFV N) (Table S3 ). A. aegypti ribosomal S7 was amplified as an internal control using primers Aag-S7 FP and Aag-S7 RP and probe Aag-S7-Probe (Table S3 ).
Renilla glo substrate
Manufactured by Promega
Sourced in United States
The Renilla-Glo substrate is a luminescent reagent used to measure the activity of the Renilla luciferase reporter gene in cell-based assays. It provides a sensitive and quantitative method for detecting Renilla luciferase expression.
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5 protocols using renilla glo substrate
1
Measuring Ren Growth in Aag2 Cells
2
Silencing RVFV Genes Using dsRNA
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dsRNA molecules targeting rMP12delNSs:hRen or eGFP were produced as described above. PCR products were generated using plasmids pTVT7-GL, pTVT7-GM, and pTVT7-GS and primers dsL-Fwd and dsL-Rev, primers dsM-Fwd and dsM-Rev, and primers dsN-Fwd and dsN-Rev, respectively (Table S3 ). The hRen dsRNA PCR template was amplified from pGL4.75 (Promega) using primers dshRen-Fwd and dshRen-Rev (Table S3 ). Transfections of Aag2 cells were performed as described above. At 4 h (p.t.), cells were infected with rMP12delNSs:hRen at an MOI of 0.1. At 18 h p.i., luciferase activity was determined using Renilla-Glo substrate (Promega) on a GloMax luminometer following cell lysis in passive lysis buffer.
3
SARS-CoV-2 Virus Infection Kinetics
4
Zika Virus Neutralizing Antibody Assay
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A Zika RVP assay (Sonnberg et al., manuscript in preparation) was used to determine neutralizing antibody titers in serum following the administration of PIZV (study days 1, 29, 57, and 71), and 30 days post-ZIKV challenge (day 101). Briefly, macaque serum was heat inactivated at 56 °C for 30 minutes and then serially diluted 3-fold in assay media for an 11-point dilution series. Diluted serum and RVP were plated in duplicate in a 384-well assay plate and incubated for 1 hour at 37 °C. Vero cells were added to each well and incubated at 37 °C for 72 hours. Renilla-Glo substrate (Promega, WI, USA) was then added to the plate and incubated for 15 minutes at room temperature. Finally, plates were analyzed by a luminometer. The effective concentration at 50% (EC50), was determined by a non-linear regression curve fit with the lower asymptote constrained to 0 in GraphPad Prism. The LLOQ for the NHP assay is 2.12 log10 EC50, below which the serum matrix interfered with the measurement. The upper limit of quantitation (ULOQ) of the standard assay is 5.0 log10 EC50. Any samples returning titers >ULOQ were retested with a higher initial pre-dilution.
5
Zika Virus Neutralization Assay
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A Zika RVP assay was also conducted as described55 (link) at Takeda Vaccines, Inc to confirm results of CDC’s R-mFRNT. Briefly, heat-inactivated serum was serially diluted and then mixed with Zika RVPs. The diluted serum and RVP mixture was then plated in duplicate in a 384-well assay plate and incubated for 1 h at 37 °C. Vero cells were added to each well and incubated at 37 °C for 72 h. Renilla-Glo substrate (Promega) was then added to the plate, incubated for 15 min at room temperature, and the plate was subsequently analyzed by a luminometer. The EC50 was determined by a non-linear regression curve fit. The serum titers indicated the final dilution level of the serum in the entire mixture of serum/RVP/Vero culture medium, while R-mFRNT (similar to traditional plaque reduction neutralization assay, PRNT) calculates the serum dilution level from serum/virus mixture only. As a result, the LOD and endpoint titer outcomes of the RVP assay are typically higher than those of R-mFRNT or PRNT.
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