T24 cells were plated overnight in T-25 flasks (5×105 cells/flask) and transiently transfected with 10 μg of ACE2-GFP, dACE2-GFP, or empty GFP-vector. After 24 hrs post-transfection, cells were pelleted and lysed with 400 μl of Lysis Buffer provided with the ACE2 activity kit (#K897, BioVision). Keeping the reaction volumes and the amount of ACE2-GFP lysates constant, we added dACE2-GFP lysate in the ratio of 0.25, 0.5 and 1.0 to ACE2-GFP, with differences in volume compensated by lysates from GFP-expressing cells. The lysate mixtures were processed in triplicates using the kit reagents and according to protocol. The carboxypeptidase activity was measured as fluorescence (Ex/Em = 365/410–460 nm) using a Promega GlowMax plate reader for two time points between 30 mins and 2 hrs after adding the corresponding substrate mix. A positive control was provided by the kit. Cell lysates were also analyzed by Western blots with C-terminal anti-ACE2 antibody (Abcam), that detects both ACE2 and dACE2, with GAPDH as a loading control.
Glowmax plate reader
Manufactured by Promega
The GlowMax plate reader is a high-performance luminescence detection system designed for sensitive and accurate measurement of luminescent signals in multi-well plates. It features a compact design and advanced optics to provide reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Ace2 activity kit, by Abcam (2 mentions)
C terminal anti ace2 antibody, by Abcam (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Psv β galactosidase plasmid, by Promega (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Operetta high content imaging system, by PerkinElmer (1 mentions)
Celltiter glo 3d cell viability assay, by Promega (1 mentions)
4 protocols using glowmax plate reader
1
Measuring ACE2 Activity in Transfected Cells
2
Investigating RPL23 and RPL29 5′ UTR Sequences
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The full-length 5′ UTR sequence of RPL23 and RPL29 containing the 5′ PES motif based on the H1 hESC CAGE database (ENCODE RIKEN CAGE) was cloned upstream of the Fluc gene. Luciferase reporters were co-transfected with a pSV-β-galactosidase plasmid (Promega) as the internal control for transfection, using Lipofectamine 3000 (Thermo Scientific). The cells were washed and harvested using Reporter lysis buffer (Promega) 24 h post transfection. Luciferase and β-galactosidase were measured using the Luciferase (Promega) and β-Galactosidase enzyme (Promega) assay systems, respectively, following the manufacturer’s instruction using a Glow-Max plate reader (Promega). The Fluc-to-β-galactosidase signal ratio was calculated for each sample to normalize for differences in transfection efficiency. Candidate-specific 5′ PES mutagenesis was performed using a Q5 site-directed mutagenesis kit (NEB).
3
Quantifying ACE2 Protease Activity
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T24 cells were transiently transfected with ACE2-GFP, dACE2-GFP, or empty GFP-vector in triplicates. After 24 hrs, cells were pelleted and lysed with 400 μl of Lysis Buffer provided with the ACE2 activity kit (#K897, BioVision). Cell lysates were analyzed by Western blots with C-terminal anti-ACE2 antibody (Abcam), and the amounts of recombinant ACE2-GFP and dACE2-GFP proteins in lysates were quantified by densitometry analysis (Imagelab, BD Biosciences) of Western blots. The amounts of ACE2 and dACE2 lysates to be used in protease assays were determined based on protein quantification. Protein lysates were processed in duplicates using the kit reagents and according to protocol. The activity was measured as fluorescence (Ex/Em = 365/410–460 nm) using Promega GlowMax plate reader for two time points between 30 mins and 2 hrs after adding the corresponding substrate mix and normalizing for baseline level. Positive control was provided by the kit.
4
3D Spheroid Viability Assay
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On the day of assemble (day 0) and days 2, 4, 6, 8, 10, 12 and 14; 12 spheroids were collected per day in order to measure the cell viability through ATP production. The ATP was quantified using a CellTiter-Glo® 3D Cell Viability Assay (Promega, cat. G9681) following the manufacturer's instructions; the luminescence was recorded in a Glow Max Plate Reader (Promega). The spheroids diameter was measured every 2 days. The images were captured with Operetta High Content Imaging System (Perkin Elmer, Waltham, MA, USA) and quantified using Harmony Software (Perkin Elmer).
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