The concentration of androstenedione in the media was measured by time-resolved fluoroimmunoassay following the methods by Yamada et al. (1997) (link). In brief, steroids were extracted from 100 μl culture medium with diethyl ether, the ether was evaporated and the resultant extracts were reconstituted in 100 μl of assay buffer (0.05 M Tris, 0.9% NaCl, 0.5% BSA, 0.05% NaN3, 0.01% Tween 40, 20 μM diethylenetriamine-N, N, N′, N″, N″-pentaacetic acid, pH 7.75). Wells of 96-well plates were coated with BSA-conjugated androstenedione prepared by a method reported elsewhere (Asahina et al., 1995 ). The steroid extracts were incubated with an antiserum against androstenedione (FKA-138, Cosmobio, Tokyo, Japan) in the coated 96-well plates. After incubation at 20 °C for 4 h, the plates were washed with PBS containing 0.05 % Tween 20 (PBST) three times. Europium (Eu)-labeled goat anti-rabbit IgG (AD0105, Perkin-Elmer, Waltham. MA) was added to each well and incubated at 20 °C for 1 h. After washes, an enhancement solution (Perkin-Elmer) was added and the fluorescence signals from dissociated Eu were measured using an Infinite F200 plate reader (TECAN, Grodig, Austria). Intra-assay CV for standard samples in the same plate were 5.56% (n = 3). For each experiment, all samples were assayed at the same time, obviating inter-assay variability.
Enhancement solution
Manufactured by PerkinElmer
Sourced in Finland, United States
The Enhancement solution is a laboratory reagent designed to optimize the performance and sensitivity of analytical instruments. It is used to improve the detection and quantification of target analytes in various sample types. The core function of this solution is to enhance the analytical signal, allowing for more accurate and reliable measurements. The specific composition and applications of the Enhancement solution may vary depending on the intended use and the requirements of the analytical method.
Automatically generated - may contain errors
Lab products found in correlation
Eu n1 labeledanti 6x his antibody, by PerkinElmer (3 mentions)
Streptavidin europium, by PerkinElmer (3 mentions)
Anti cd69 clone h1.2f3, by BioLegend (2 mentions)
Anti cd25, by BioLegend (2 mentions)
Anti cd3ε fitc clone 145.2c11, by BioLegend (2 mentions)
Anti cd4 percp clone rm4 5, by BioLegend (2 mentions)
Victor 1420 multilabel counter, by PerkinElmer (2 mentions)
Streptavidin coated plates, by PerkinElmer (2 mentions)
Infinite f200 plate reader, by Tecan (1 mentions)
Infinite m1000, by Tecan (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Multicalc software, by PerkinElmer (1 mentions)
Protamine sulfate, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
Envision multiplate reader, by PerkinElmer (1 mentions)
Wallac victor3, by PerkinElmer (1 mentions)
Victor3 multilabel counter, by PerkinElmer (1 mentions)
Prism 10, by GraphPad (1 mentions)
Delfia assay buffer, by PerkinElmer (1 mentions)
Low molecular weight heparin, by Merck Group (1 mentions)
15 protocols using enhancement solution
1
Androstenedione Quantification by TRFIA
2
DQ0602 Peptide Binding Assay
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Peptide binding assay was conducted as previously described81 (link),82 (link). Briefly, DQ0602 was incubated with biotinylated EBV epitope (Bio-EBV486–500, Bio-(GGG)RALLARSHVERTTDE) and RFX4 peptide for 72 h at 37 °C, followed by incubation with monoclonal anti-DQ (SPV-L3) antibody (Cat# BNUM0200-50, Biotium) in a high binding 96-well plate (REF# 9018, Corning). DELFIA time-resolved fluorescence (TRF) intensity was detected using a Tecan Infinite M1000 after successive incubation with Europium (Eu)-labelled streptavidin (Cat# 1244-360, PerkinElmer) and enhancement solution (Cat# 1244-105, 9 PerkinElmer) (Supplemental Fig. S7 A). Plate was washed 5 times with 300 µl/well wash buffer (0.05% Tween-20 in PBS) to remove nonspecific binding. Bio-EBV486-500 alone is as the reference (positive control) and without any peptide is as negative control. Compared with the reference, RFX4 peptides with lower than 25% and 25–50% of fluorescence are considered as strong and weak peptides, respectively.
3
T-cell Activation and Proliferation Assay
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Cell-free co-culture supernatants were harvested at the indicated times. IL-2 and IFNγ were determined by immunoassay. Antibody pairs were purchased from Biolegend. Cytokine levels were determined using streptavidin-europium and enhancement solution (both from Perkin Elmer) and detected on a Victor 1420 multilabel counter (Perkin Elmer). Day 1 or 6 BMDC and OT-II T-cell co-cultures were harvested and stained with anti-CD3ε-FITC (clone 145.2C11; Biolegend), anti-CD4-PerCP (clone RM4-5; Biolegend), anti-CD69 (clone H1.2F3; Biolegend), anti-CD25 (PC61; Biolegend) and Fixable Viability Dye eFluor 506. Cells were fixed in 1% PFA in PBS. Proliferating and activated T-cell populations were determined gating on live, singlet, CD3+, CD4+ cells and by CTV dilution.
4
Measuring HPSE Activity in MM Cells
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HPSE action was measured by an ELISA-like method using HS biotinylated, as previously described.29 (link) Transfected MM cells (1×105 per well) were cultured for 3 days in 60-mm plates. The cells were collected by centrifugation and resuspended in 500 μL of sodium acetate 25 mM, pH 5.0, containing protease inhibitors (ThermoFisher Scientific, USA), and 50 μL of cell extract was incubated with the pre-coated plate, revealed with europium-labeled streptavidin, washed and submitted to 200 μL of enhancement solution (PerkinElmer Life Sciences-Wallac Oy, Finland). Free europium was measured and the data analyzed in the MultiCalc software (PerkinElmer Life Sciences-Wallac Oy, Finland). The product obtained by HPSE incubation was expressed by the ratio of degraded HS and total protein from the cellular fraction (μg of HS/μg of total protein).
5
Measurement of Heparanase Activity
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AGS and MKN45 gastric cells were treated with RSV (5–25 μM) or ethanol 1% for 24 h. Heparanase activity was measured using an ELISA-like assay using biotinylated heparan sulfate [56 (link)]. Briefly, the 96-well plate (Nalge Nunc, Rochester, NY, USA) was incubated with 200 µL of protamine sulfate (Sigma-Aldrich, St. Louis, MO, USA, 10 µg/mL) overnight at 37 °C. Subsequently, 100 µL of biotinylated heparan sulfate (10 µg/mL) was immobilized for 18 h at 37 °C. The enzymatic assay was performed in sodium acetate 25 mM, pH 5.5, in a final volume of 100 µL. A volume of 50 μL of the cellular extract was incubated with the pre-coated 96-well plate with biotinylated heparan sulfate. The immobilized biotinylated heparan sulfate not digested by heparanase was bound to streptavidin conjugated with europium. Then, an enhancement solution (200 μL) (PerkinElmer Life Sciences) was added to release the europium. Free europium was measured, and the data were analyzed in the MultiCalc software v2.7 (PerkinElmer Life Sciences-Wallac Oy, Turku, Finland). The product obtained via HPSE1 was expressed by the ratio of degraded heparan sulfate (HS) and total protein from the cellular fraction (μg degraded HS/μg total protein). The values were expressed as a percentage of heparanase activity.
6
Biotinylated-BH3 Peptide Binding Assay
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Each well of 96-well streptavidin-coated plates (PerkinElmer) was
incubated with 100 μL of a 600 ng/mL biotinylated-BH3 peptide
of sequence biotin-amino-hexanoic acid-EDIIRNIARHLAQVGDSMDR-NH2 for 2 h and washed 3 times with a wash solution (PerkinElmer).
Subsequently, a mixture containing 11 μL of protein and a serial
dilution of the test compounds as well as 89 μL of an Eu-N1-labeled
anti-6x-His antibody (PerkinElmer) was added to each well and incubated
for 2 h. Plates were then washed 3 times, and 200 μL of enhancement
solution (PerkinElmer) was added to each well. After a 10 min incubation,
fluorescence measurements were taken with a Victor X5 microplate reader
with the excitation and emission wavelengths of 340 and 615, respectively.
All the incubations were performed at room temperature. The final
protein concentrations used for hBfl-1 and hMcl-1 were 15 and 16 nM,
respectively and the final anti-6x-His antibody used was 22.2 ng/well.
Fluorescence readings were normalized to those of 1% DMSO-treated
wells and reported as % inhibition. Prism 9 (GraphPad) was used to
calculate IC50 values.
incubated with 100 μL of a 600 ng/mL biotinylated-BH3 peptide
of sequence biotin-amino-hexanoic acid-EDIIRNIARHLAQVGDSMDR-NH2 for 2 h and washed 3 times with a wash solution (PerkinElmer).
Subsequently, a mixture containing 11 μL of protein and a serial
dilution of the test compounds as well as 89 μL of an Eu-N1-labeled
anti-6x-His antibody (PerkinElmer) was added to each well and incubated
for 2 h. Plates were then washed 3 times, and 200 μL of enhancement
solution (PerkinElmer) was added to each well. After a 10 min incubation,
fluorescence measurements were taken with a Victor X5 microplate reader
with the excitation and emission wavelengths of 340 and 615, respectively.
All the incubations were performed at room temperature. The final
protein concentrations used for hBfl-1 and hMcl-1 were 15 and 16 nM,
respectively and the final anti-6x-His antibody used was 22.2 ng/well.
Fluorescence readings were normalized to those of 1% DMSO-treated
wells and reported as % inhibition. Prism 9 (GraphPad) was used to
calculate IC50 values.
7
Autoantibody Detection in Patient Sera
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Detection of autoantibody IgG titers in patient sera was performed using pre-coated plates (ANAcombi: RNP-70, Sm, RNP/Sm, SS-A, SS-B, Scl-70, Centromer B, Jo-1; Orgentec, Mainz, Germany) or Poly-L-lysine pre-coated maxisorp black plates (Thermo Fisher) for dsDNA. Patient or HV sera were pre-diluted (1:250 or 1:50 with serial dilutions for dsDNA including reference controls) with reagent buffer (1% BSA, 0.05% v/v Tween 20 in PBS) in 50 μL and added to plates. After 2 h on an orbital shaker, plates were washed (PBS/0.05% v/v Tween 20) and biotinylated anti-human IgG (1:5000, 50 μL/well) was added for 2 h. Plates were washed four times prior to addition of Streptavidin-Eu antibody (1:1000, 50 μL/well, PerkinElmer) for 1 h. After washing, plates were analyzed using an EnVision Multiplate reader following addition of Enhancement solution (PerkinElmer) for 15 min in the dark. Qualitative titers of each serum were calculated according to the manufacturer’s protocol (Orgentec) with scoring index: negative <1.0, borderline 1.0–1.2 and positive >1.2. For dsDNA IgG titers, quantification based on scoring: negative <20 IU/mL, borderline 21–100 IU/mL and positive >100 IU/mL (IgG).
8
Amyloid-like Fibrils Binding Assay
9
Europium-Based Cytotoxicity Assay Protocol
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5–10 × 106 target cells were labeled with europium for 20 min at 4°C using a standard published procedure50 (link). 104 europium-labeled targets and serial dilutions of effector cells at varying E:T ratio or at fixed E:T ratio as indicated in the figure legends were incubated in 200 μl of CTL medium (CTL stimulation medium with no penicillin-streptomycin) in 96-well V-bottom plates. The plates were centrifuged at 500 × g for 3 min and incubated at 37°C for 4 hours. 50 μl of the supernatant was harvested and added to 150 μl of enhancement solution (Perkin-Elmer) in 96-well flat-bottom plates and europium release was measured by time resolved fluorescence using the VICTOR3 Multilabel Counter (Perkin-Elmer).
Specific cytotoxic activity was determined using the formula: % specific release = [(experimental release - spontaneous release)/(total release - spontaneous release)] × 100. Spontaneous release of the target cells was less than 25% of total release by detergent in all assays for the results of the assay to be valid. Spontaneous release of the target cells was determined by incubating target cells in medium without T cells. Non-specific lysis was determined by measuring the lysis of target cells loaded with control peptides (C). All assays were done in triplicates and repeated 2–3 times to confirm data.
Specific cytotoxic activity was determined using the formula: % specific release = [(experimental release - spontaneous release)/(total release - spontaneous release)] × 100. Spontaneous release of the target cells was less than 25% of total release by detergent in all assays for the results of the assay to be valid. Spontaneous release of the target cells was determined by incubating target cells in medium without T cells. Non-specific lysis was determined by measuring the lysis of target cells loaded with control peptides (C). All assays were done in triplicates and repeated 2–3 times to confirm data.
10
Quantifying Protein-Peptide Interactions
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A solution containing 600
ng/mL of biotinylated-BH3 peptide (biotin-aminohexanoic acid-EDIIRNIARHLAQVGDSMDR-NH2) was added to each well of 96-well streptavidin-coated plates
(PerkinElmer). At the end of a 2 h incubation, plates were washed
3 times. Subsequently, a solution of 6His-tagged-hMcl-1(172–323)
or 6His-tagged-hBfl-1(1–149) protein and the test peptides
were preincubated for 2 h and added to the washed streptavidin-coated
plates along with a Eu–N1-labeled anti-6xHis antibody (PerkinElmer,
1:2000) solution and further incubated for 2 h on a microplate shaker.
Plates were then washed 3 times, and each well was incubated with
200 μL of the enhancement solution (PerkinElmer) for 10 min.
Fluorescent readings (VICTOR X5 microplate reader) were then measured
by using the excitation and emission wavelengths of 340 and 615 nm,
respectively. Fluorescent counts were normalized to those of DMSO
wells and reported as % inhibition, and Prism 10 (GraphPad) was used
to calculate the IC50 values. Protein, peptide, and antibody
solutions were prepared in a DELFIA assay buffer (PerkinElmer), and
all of the incubations were performed at room temperature. Each well
received a final DMSO concentration of 1%. The final concentrations
of 6His-tagged-hMcl-1(172–323) and 6His-tagged-hBfl-1(1–149)
used were 16 and 15 nM, respectively.
ng/mL of biotinylated-BH3 peptide (biotin-aminohexanoic acid-EDIIRNIARHLAQVGDSMDR-NH2) was added to each well of 96-well streptavidin-coated plates
(PerkinElmer). At the end of a 2 h incubation, plates were washed
3 times. Subsequently, a solution of 6His-tagged-hMcl-1(172–323)
or 6His-tagged-hBfl-1(1–149) protein and the test peptides
were preincubated for 2 h and added to the washed streptavidin-coated
plates along with a Eu–N1-labeled anti-6xHis antibody (PerkinElmer,
1:2000) solution and further incubated for 2 h on a microplate shaker.
Plates were then washed 3 times, and each well was incubated with
200 μL of the enhancement solution (PerkinElmer) for 10 min.
Fluorescent readings (VICTOR X5 microplate reader) were then measured
by using the excitation and emission wavelengths of 340 and 615 nm,
respectively. Fluorescent counts were normalized to those of DMSO
wells and reported as % inhibition, and Prism 10 (GraphPad) was used
to calculate the IC50 values. Protein, peptide, and antibody
solutions were prepared in a DELFIA assay buffer (PerkinElmer), and
all of the incubations were performed at room temperature. Each well
received a final DMSO concentration of 1%. The final concentrations
of 6His-tagged-hMcl-1(172–323) and 6His-tagged-hBfl-1(1–149)
used were 16 and 15 nM, respectively.
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