Two days post transfection, cells plated on clear poly L-lysine coated 96 well plates were washed with PBS and fixed with either methanol (for total expression) or 4% paraformaldehyde (for surface expression). Cells were then blocked with 1% milk and incubated in peroxidase conjugated anti-HA antibody. The plate was washed with TBS-T three times and then incubated with 100 μL 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate (Sigma, St. Louis, MO, USA) for 30 minutes. 100 μL of 1 mol/L sulfuric acid was added to each well to stop the reaction. Absorbance was then read at 450 nm on a Spectramax M3 plate reader (Molecular Devices). The absorbance from untransfected cells was subtracted and then the cell surface labeled signal was plotted as a percentage of total signal (calculated as the non-permeabilized signal divided by the permeabilized signal ×100) and plotted. Total expression as a percentage of wild-type HA-MC1R for each experiment was also plotted. Data shown is from 3 independent experiments (mean ± SEM). Significant differences from wild-type were determined using one-way ANOVA with Dunnet’s post-hoc.
3 3 5 5 tetramethylbenzidine liquid substrate
Manufactured by Merck Group
Sourced in United States
3,3′,5,5′-tetramethylbenzidine liquid substrate is a chromogenic substrate used in various immunoassay and enzymatic detection applications. It serves as a sensitive substrate for the detection and quantification of peroxidase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m3, by Molecular Devices (2 mentions)
Phytohemaglutinin, by Merck Group (1 mentions)
Biotinylated detection antibody, by Mabtech (1 mentions)
Streptavidin horseradish peroxidase conjugate in pbs bsa 0.5, by Agilent Technologies (1 mentions)
Elispot plate, by Merck Group (1 mentions)
Automated elisa reader, by Tecan (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg antibody, by Abcam (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Goat anti mouse igm, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Elisa plate reader, by Molecular Devices (1 mentions)
Immulon 4hbx plate, by Thermo Fisher Scientific (1 mentions)
Powerwave 340 microplate reader, by Agilent Technologies (1 mentions)
Varioskan flash microplate reader, by Thermo Fisher Scientific (1 mentions)
Stop reagent, by Merck Group (1 mentions)
Immulon 4hbx, by Thermo Fisher Scientific (1 mentions)
Pherastar fs, by BMG Labtech (1 mentions)
12 protocols using 3 3 5 5 tetramethylbenzidine liquid substrate
1
Quantifying Cell Surface Expression of HA-MC1R
2
Quantifying Cytokine-Producing Cells Using ELISPOT
3
Antibody Binding Assay for VLPs
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Wells of 96-well microplates were coated with wild-type or mutant VLPs or pentamers. After plate blocking (300 ng/well, incubated overnight at 4°C), 2-fold serial dilutions of the antibody were added to the wells and incubated for 1 h at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (diluted 1:5,000 in PBS; Abcam, Inc.; Cambridge, United Kingdom) was used to detect the antibody titers, followed by 50 µl of 3,3′,5,5′-tetramethylbenzidine liquid substrate (Sigma-Aldrich, St. Louis, MO) per well for 30 min at 37°C. The absorbance at 450 nm (reference, 620 nm) was recorded using an automated ELISA reader (Tecan, Männedorf, Switzerland). Endpoint titers were defined as the highest plasma dilution that resulted in an absorbance value 2 times higher than that of nonimmune plasma with a cutoff value of 0.05. Data are presented as log10 values. The median effective concentration (EC50 [nanograms per milliliter]) is defined as the antibody concentration for achieving 50% binding with the antigen.
4
T Cell-Dependent Antigen Immune Response
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Mice were stimulated with T cell-dependent antigen TNP-BSA, a 2,4,6-trinitrophenyl hapten conjugated with bovine serum albumin (50 μg per mice), solved in complete Freund's adjuvant (Sigma-Aldrich, Diegem, Belgium) by subcutaneous injection. The blood was collected by eye puncture before stimulation and on days 7, 10, 14, 21, and 28 poststimulation. On day 28, the mice underwent for a second time stimulation with TNP-BSA and blood samples were taken on days 35 and 42. Analysis of anti-TNP IgM and IgG in the collected sera was done by ELISA procedure in prepared 96-well ELISA plates (coated overnight at 4°C with 5 μg/ml TNP-BSA in phosphate-buffered saline (PBS)) with sheep anti-mouse IgG (H/L) : horse radish peroxidase (HRP) and with goat anti-mouse IgM : HRP (Bio-Rad AbD Serotec, Oxford, United Kingdom), respectively. Peroxidase activity was detected by adding 3,3′,5,5′-tetramethylbenzidine liquid substrate (Sigma-Aldrich, Diegem, Belgium). Reaction was stopped by adding 1 M HCl. Optical absorbance was measured by the ELISA reader at 450 nM.
5
Quantifying Cell Surface Expression of HA-MC1R
6
Sandwich ELISA for IL-1α Quantification
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We developed an IL-1α sandwich ELISA by using the anti-IL-1α mAb (1 μg/ml) as a capture antibody and affinity-purified rabbit anti-IL-1α polyclonal antibody (0.1 μg/ml) as a detection antibody. After washing, the wells were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) for 1 h and then developed with 3,3′,5,5′-tetramethylbenzidine liquid substrate (Sigma-Aldrich). The absorbance at 450 nm was measured using an ELISA plate reader (Molecular Devices, Sunnyvale, CA).
7
CHIKV Antibody Quantification by ELISA
8
Quantitative ELISA for IgG Antibodies
9
Cytokine Profiling of Polarized Monocytes
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PB monocytes (105 cells/well) were incubated for 72 h with polarizing stimuli diluted in culture medium containing 5% FCS and subsequently stimulated with LPS (from E. coli 0111: B4, Sigma-Aldrich). After 4 h (for TNF) or 24 h for (IL1β and IL6), culture supernatant fractions were collected and assayed for cytokine production with OptEIA ELISA, following the manufacturer’s instructions (BD Biosciences). In experiments using THP1 macrophages treated with siRNA targeting ID3 expression, cells were siRNA-transfected and allowed to differentiate (see below). They were then stimulated with LPS (5 × 104 cells/well) and assayed for cytokine production as above. Color was developed by adding 3,3′,5,5′-tetramethylbenzidine liquid substrate (Sigma-Aldrich). Optical density was read at 450 nm on a Varioskan Flash microplate reader (ThermoFisher Scientific Inc.).
10
Quantifying Surface Expression of HA-tagged Proteins
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Approximately 10,000 HA tagged construct transfected cells were added to wells of a poly-L-lysine coated 96 well plate. The following day, cells were washed with PBS and fixed with either methanol (for total expression) or 4% paraformaldehyde (for surface expression). Cells were then blocked with 1% milk and incubated in peroxidase conjugated anti-HA antibody. The plate was washed with TBS-T three times and then incubated with 100 µL 3,3′,5,5′-Tetramethylbenzidine Liquid Substrate (Sigma, St. Louis, MO, USA) for 30 minutes. 100 µL of 1 mol/L sulfuric acid was added to each well to stop the reaction. Absorbance was then read at 450 nm on a Spectramax 250 plate reader. The absorbance from untransfected cells was first subtracted and then the cell surface labeled signal was plotted as a percentage of total signal (calculated as the non-permeabilized signal divided by the permeabilized signal ×100) and then normalized to wild-type HA-MC4R expression for that experiment. Significant differences from wild-type were determined using one way ANOVA with Dunnet's post-hoc.
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