The cytotoxic activity of Jurkat cells expressing CD16 (FcγRIIIA-158V) and a NFAT luciferase reporter on RSV-infected HEp-2 cells was measured using a bioluminescent ADCC reporter assay (Promega G7010). HEp-2 target cells were prepared by seeding 25,000 cells per well in 96-well plates. Cells were plated with RSV A000, (i.e., a recombinant RSV resembling the contemporary RSV A sequence (42 (link))) at a multiplicity of infection of 1 and incubated for 24 hours at 37°C and 5% CO2. Either purified antibody or clinical samples were then added to the plates along with 75,000 Jurkat effector cells per well following manufacturer kit recommendations. Plates were incubated for 6 hours at 37°C and 5% CO2. Bio-Glo™ luciferase reagent was reconstituted following manufacturer recommendations (Promega G7010) and then added to the plates. Plates were incubated at RT in the dark for 30 minutes. Luminescence was read on an EnVision plate reader.
Bio glo luciferase reagent
Manufactured by Promega
Bio-Glo luciferase reagent is a luminescent reagent used to detect and quantify luciferase reporter gene expression. The reagent contains the necessary components for the luciferase-catalyzed oxidation of the substrate luciferin, resulting in the emission of light. This light output can be measured using luminometers or other compatible detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
G7010, by Promega (1 mentions)
96 well plate, by GenScript (1 mentions)
White bottom 96 well plate, by Corning (1 mentions)
Adcc reporter bioassay kit, by Promega (1 mentions)
Low igg fbs, by Promega (1 mentions)
Synergy 4, by Agilent Technologies (1 mentions)
4 protocols using bio glo luciferase reagent
1
Cytotoxicity Assay of RSV-Infected Cells
2
SARS-CoV-2 Spike Protein Pseudovirus Neutralization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse serum was blinded and diluted in a duplicate fourfold series from 1:5 to 1:81,920. Serum was mixed 1:1 with pseudovirus containing the SARS-CoV-2 spike protein and a luciferase reporter in a 96-well plate (GenScript). After 1 hour of incubation at room temperature, 20,000 human embryonic kidney (HEK) 293 cells overexpressing Ace2 were added to each well. Cells and pseudovirus were incubated at 37°C, 5% CO2 for 48 hours, after which culture medium was removed and Bio-Glo luciferase reagent (Promega) was added to the wells. Luciferase signal was measured using an EnVision plate reader, and % inhibition was calculated using the following formula where X is the luciferase signal, min is the average signal from duplicate wells without pseudovirus, and max is the average signal from duplicate wells without serum. Log ID50 values were calculated in the same manner described for the RBD/Ace2-blocking assay.
3
ADCC Assay for Monoclonal Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ADCC capacity of each MAb was measured using an ADCC Reporter Bioassay kit (Promega) largely in line with the manufacturer’s instructions. In brief, 3 × 104 Vero.E6 cells per well were seeded in white-bottom 96-well plates (Corning) and infected with VSV-ANDV in MEM at an MOI of 1 16 h later. After 2 days of incubation with virus at 37°C, 3-fold serial dilutions of each MAb were prepared in MEM, starting with 90 μg/ml. A negative-control antibody (KL-2G12) was used as well for determining the background. These MAbs were then added to the infected plate with effector cells from the Promega kit at a ratio of 3:2 (effector cells to infected cells) and additional medium such that the effective starting concentration of each MAb was 30 μg/ml. The plates were then incubated at 37°C for 6 h. Bio-Glo Luciferase reagent (Promega) was then added, and luminescence was measured immediately.
4
ADCC/ADCP Bioassay for NDV-HexaPro-S Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vero.E6 cells (ref) were seeded in 96 well plates (Corning; 3340) at a seeding density of 2x104 cells per well 24 hours prior to infection with NDV-HexaPro-S82 (link) at a multiplicity of infection (MOI) of 2. One hour later the inoculum was removed and replaced with 100μl of Roswell Park Memorial Institute (RPMI-1640) media (Thermofisher). 48 hours later the media was removed and replaced with 25 μL of RMPI-1640 media supplemented with 2% low-IgG FBS (Promega). Antibodies were serially diluted 1:10 in RPMI 1640 media from a starting concentration of 30 μg/mL to a final concentration of 0.014 μg/mL and 25 μL of antibody dilution was added to the plate. CR9114 was utilized as a negative control48 (link). MAb dilutions were not added to 16 wells per plate to provide background luminescence readings. 3x106 ADCC and ADCP Bioassay Effector Cells expressing the human FcγRIIIa or FcγRIIa receptors (Promega) were added per well and the cells were incubated in at 37°C with 5% CO2 for 6 hours. 75 μL of Bio-Glo luciferase reagent (Promega) was then added to each well and the luminescence was read in a Synergy 4 (BioTek) plate reader. The background luminescence reading was used to calculate AUC using GraphPad Prism 10.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!