Supernatants were collected at 48 hrs from lymphocytes incubated with PLP139–151 peptide, and stored at −80°C until tested. Flat-bottomed 96-well plates (Corning) were coated with anti-IL-2 capture antibody (BD Bioscience, San Jose, CA, USA) overnight at 4°C. Plates were washed, incubated with supernatants, washed and incubated with biotinylated secondary antibody (BD Bioscience). Europium streptavidin (PerkinElmer) was added and plates were developed by using the Delfia Enhancement solution (PerkinElmer). Fluorescence was measured using a Wallac Victor 2 Multi-label Counter (PerkinElmer). Experimental values were determined through the use of a standard curve derived from serial dilutions of a known quantity of recombinant IL-2.
Streptavidin europium
Manufactured by PerkinElmer
Sourced in United States, Finland
Streptavidin-europium is a laboratory reagent used in fluorescence-based assays. It consists of the protein streptavidin, which has a high affinity for the small molecule biotin, conjugated to the fluorescent lanthanide chelate europium. This conjugate can be used to detect and quantify biotinylated molecules, such as proteins, nucleic acids, and other biomolecules, in various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Enhancement solution, 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)
Heparan sulfate, by Merck Group (2 mentions)
Del a enhancement solution, by PerkinElmer (2 mentions)
Protamine sulfate, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Biotinylated secondary antibody, by BD (1 mentions)
Wallac victor 2 multi label counter, by PerkinElmer (1 mentions)
Delfia enhancement solution, by PerkinElmer (1 mentions)
96 well flat bottomed plate, by Corning (1 mentions)
Wallac victor2, by PerkinElmer (1 mentions)
Low molecular weight heparin, by Merck Group (1 mentions)
Biotinylated goat anti mouse igg, by Merck Group (1 mentions)
Wallac victor3 plate reader, by PerkinElmer (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
96 well polystyrene microplate, by Corning (1 mentions)
7 protocols using streptavidin europium
1
Quantification of IL-2 in Lymphocyte Supernatants
2
Quantitative Peptide-Binding Assays
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Peptide-binding assays were performed, as previously described [13 (link)]. In short, cell lysates from HLA class II homozygous B-lymphoblastoid cell lines were incubated on SPV-L3- (anti-HLA-DQ)- or B8.11.2- (anti-HLA-DR)-coated (10 μg/ml) FluoroNunc 96-well plates at 4 °C overnight. Test peptides in the range of 0 to 300 μM were mixed with a fixed concentration (0.6 μM) of biotinylated indicator peptide and added to the wells. Bound indicator peptide was detected using Europium-streptavidin (PerkinElmer, Boston, MA, USA) and measured in a time-resolved fluorometer (PerkinElmer, Wallac Victor2). IC50 values were calculated based upon the observed binding of the test peptide against the fixed concentration indicator peptide. The IC50 value depicts the concentration of test peptide required for a loss of 50 % of the indicator peptide signal. IC50 values greater than 300 μM were classified as non-detectable binding.
3
Multivalent Binding Assay for Amyloid Detection
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Bioactivity of the Fcp5, peptide p5, and Fc2a control was assessed using a EuLISA. The wells of a 96-well polystyrene microplate (Corning, Corning, NY, USA) were coated either with poly-l -lysine (Sigma-Aldrich) followed by low molecular weight heparin (Sigma-Aldrich), amyloid-like fibrils (0.83 µM), or monomeric forms of rVλ6Wil, Aβ(1–40) or human IAPP(Ile26Pro). Wells coated with fibrils or monomeric proteins were incubated overnight at 37 or 4°C, respectively. The wells were then treated with 200 µL of blocking buffer (PBS containing 1% bovine serum albumin; BSA) for 1 h at room temperature before washing with PBS and addition of the appropriate concentration of Fcp5, biotinylated-p5, or Fc2a in PBS with 1% (w/v) BSA and 0.05% (v/v) tween 20. Following a wash step, the bound Fcp5 and Fc2a were detected by addition of biotinylated goat anti-mouse IgG (Sigma-Aldrich). After washing with PBS/tween, wells were incubated with 100 µL of europium–streptavidin (Perkin Elmer, Waltham, MA, USA) and washed, followed by addition of 100 µL enhancement solution (Perkin Elmer) before measurement of time-resolved fluorescence emission using a Wallac Victor 3 plate reader (Perkin Elmer).
4
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.
5
Cytokine Analysis of BMDC-OT-II T-cell Co-culture
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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(eBiosciences). Cells were fixed in 1% PFA in PBS. Proliferating and activated T-cell populations were determined by gating on live, singlet, CD3+, CD4+ cells and by CTV dilution.
6
Heparanase Activity Quantification
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The cellular fraction used to perform the enzymatic assay was scraped from culture plates with sodium acetate buffer, pH 5.5, and protease inhibitor cocktail (Sigma-Aldrich). Total cell protein was estimated by Pierce™ Coomassie Plus (Bradford) Assay Kit (Thermo Fisher Scienti c), as described by the manufacturer. It is important to point out that the values that express heparanase activity were adjusted by the total protein of cellular extract, to eliminate bias due to some possible anti-proliferative or proproliferative effect. Enzymatic activity of heparanase was measured using 1 µg heparan sulfate 15% biotinylated (bovine kidney, Sigma-Aldrich) immobilized on a 96-well plate previous coated with protamine sulfate (Sigma-Aldrich), following the instructions described by Bouças and coworkers (33) . Subsequently, the cellular fraction (15 µg of total protein) was added to the plate containing immobilized biotinylated heparan sulfate and maintained for 4 hours at 37°C, followed by streptavidin-europium incubation (Perkin-Elmer, Finland), for 40 minutes, at room temperature. Finally, a Del a® enhancement solution (Perkin-Elmer) was added and uorescence was quanti ed following the protocol described by Melo et al (34) .
7
Heparanase Activity Quantification
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The cellular fraction used to perform the enzymatic assay was scraped from culture plates with sodium acetate buffer, pH 5.5, and protease inhibitor cocktail (Sigma-Aldrich). Total cell protein was estimated by Pierce™ Coomassie Plus (Bradford) Assay Kit (Thermo Fisher Scienti c), as described by the manufacturer. It is important to point out that the values that express heparanase activity were adjusted by the total protein of cellular extract, to eliminate bias due to some possible anti-proliferative or proproliferative effect. Enzymatic activity of heparanase was measured using 1 µg heparan sulfate 15% biotinylated (bovine kidney, Sigma-Aldrich) immobilized on a 96-well plate previous coated with protamine sulfate (Sigma-Aldrich), following the instructions described by Bouças and coworkers (33) . Subsequently, the cellular fraction (15 µg of total protein) was added to the plate containing immobilized biotinylated heparan sulfate and maintained for 4 hours at 37°C, followed by streptavidin-europium incubation (Perkin-Elmer, Finland), for 40 minutes, at room temperature. Finally, a Del a® enhancement solution (Perkin-Elmer) was added and uorescence was quanti ed following the protocol described by Melo et al (34) .
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