Mice were anesthetized by isoflurane inhalation and then bled retroorbitally using heparinized micro-hematocrit capillary tubes (Fisherbrand). Twenty to fifty μl of blood were collected in 1 ml eppendorff tubes and centrifuged for 10 min at 10,000 rpm to separate the serum. Serum immunoglobulin titers were measured by ELISA. Polystyrene microtiter plates (Nunc) were coated overnight with 50 μg/ml of ovalbumin (OVA) protein (Sigma), then washed and blocked with bovine serum albumin (1%). Serum samples were applied at 1:5 dilution, and incubated for 3 hours at room temperature, then washed and incubated with alkaline-phosphatase-goat-anti-mouse IgM, IgA, IgG1 (1:100) developed by the addition of p-nitrophenyl phosphate solution (Sigma-Aldrich). Optical density (OD) 490 nm was measured using a Molecular Devices spectrophotometer.
Polystyrene microtiter plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
Polystyrene microtiter plates are a type of laboratory equipment used for various applications. They consist of a flat surface with multiple small wells or depressions, typically made of polystyrene material. The plates are designed to hold small volumes of liquids or samples, enabling multiple experiments or tests to be conducted simultaneously in a compact format.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by Dojindo Laboratories (3 mentions)
P nitrophenyl phosphate, by Merck Group (3 mentions)
Microhematocrit capillary tube, by Thermo Fisher Scientific (2 mentions)
Ovalbumin ova protein, by Merck Group (2 mentions)
P nitrophenyl phosphate solution, by Merck Group (2 mentions)
Calf thymus histone h3, by Roche (2 mentions)
Model 680, by Bio-Rad (2 mentions)
Recombinant cytokines, by Thermo Fisher Scientific (2 mentions)
Anti mouse il 17 clones tc11 18h10 tc, by BD (2 mentions)
Ifn γ an 18 r4 6a2, by BD (2 mentions)
Versamax, by Molecular Devices (2 mentions)
680 microplate reader, by Bio-Rad (1 mentions)
Original multiskan ex, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Bio-Rad (1 mentions)
Tween 20, by Merck Group (1 mentions)
Alkaline phosphatase, by Merck Group (1 mentions)
Human mbdnf, by Alomone (1 mentions)
Elisa reader, by Agilent Technologies (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Methyl alcohol, by Merck Group (1 mentions)
25 protocols using polystyrene microtiter plate
1
Serum Immunoglobulin Quantification by ELISA
2
Quantification of Syndecan-1 in Plasma
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Polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μL/well of anti-syndecan-1 polyclonal antibody (R&D Systems, Minneapolis, MN, USA) in phosphate-buffered saline (PBS), and incubated overnight at 37°C. After three washes with PBS containing 0.05% Tween-20, the remaining binding sites were blocked by incubation with 400 μL/well of PBS containing 1% bovine serum albumin (BSA) for 2 h. The plates were washed again, and incubated with 100 μL/well of diluted standard (recombinant syndecan-1, R&D Systems) and plasma samples (1:10 dilution in 0.2 mol/L Tris pH 7.4, 0.15 mol/L NaCl, and 1% BSA) for 24 h at room temperature. After washing, the plates were incubated with 100 μL/well of peroxidase-conjugated anti-syndecan-1 monoclonal antibody (R&D Systems) for 2 h at room temperature. The plates were washed again, and incubated with the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (Dojindo Laboratories, Kumamoto, Japan) for 30 min at room temperature. The reaction was terminated by addition of 0.35 mol/L Na2SO4, and the absorbance at 450 nm was measured with a 680 microplate reader (Bio-Rad, Hercules, CA, USA). The lower detection limit of the assay was 0.5 ng/mL, and linearity was observed in the range up to 50 ng/mL. The intra-assay and inter-assay coefficients of variation were 4.5–7.1% and 3.1–9.5%, respectively, at 12.5–25 ng/mL.
3
Quantifying Vibrio vulnificus Biofilm Formation
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To quantify the biofilms of the V. vulnificus strains, each well of 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark) was inoculated with 200 μl of culture diluted to an A600 of 0.05 in VFMG supplemented with 0.01% arabinose. After static incubation at 30°C for different times, supernatants were removed from the wells, and the remaining biofilms were stained with 1% (w/v) crystal violet solution for 15 min. The stained biofilms were quantified by the elution of the crystal violet with ethanol and measurement of absorbance at 570 nm (A570) as described previously (Ko and Choi, 2021 (link)).
4
Serum Histone H3 Quantification
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Serum histone H3 levels were measured as recently described [9 (link)]. Briefly, polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μL of 1 mg/L anti-histone H3 antibody (Shino-Test Corporation, Sagamihara, Japan). After washing and blocking, the plates were incubated with 100 μL of diluted calibrator and serum samples for 24 h at room temperature. After washing, the plates were incubated with 100 μL of anti-histone H3 peroxidase-conjugated antibody (Shino-Test Corporation) for 2 h at room temperature. The plates were washed again, and incubated with the chromogenic substrate 3,3′,5,5′-tetra-methylbenzidine (Dojindo Laboratories, Kumamoto, Japan). The reaction was terminated with 0.35 mol/L Na2SO4, and the absorbance at 450 nm was measured with a microplate reader (Model 680; Bio-Rad, Hercules, CA). A standard curve was obtained with purified calf thymus histone H3 (Roche, Stockholm, Sweden). The lower detection limit of the ELISA was 2 ng/mL, and linearity was observed in the range up to 250 ng/mL. The ELISA specifically detected histone H3, and did not react with other histone family proteins, including histone H2A, H2B and H4 [9 (link)].
5
ELISA for Native and AGE-Modified Thyroglobulin
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ELISA was performed as previously described [17 (link), 18 (link)]. Polystyrene microtiter plates (Nunc) were divided in two sections: the first half was coated with 10 μg/mL of native Tg in 100 μL of carbonate-bicarbonate buffer 0.1 M, pH 9.6 and the other half with the same concentration of AGE-Tg in the same buffer and incubated for 1 h at 37°C and then overnight at 4°C. Wells were washed with PBS, followed by blocking with 5% bovine serum (BS) in PBS (PBS-BS) for 1 h at 37°C. Each coated antigen was incubated with the same serum samples, added in threefold dilutions (1/50 to 1/1350) in PBS-BS-T 0.1% for 2 h, followed by the addition of alkaline phosphatase-conjugated goat anti-human IgG (γ-chain specific) (250 ng/mL) (Sigma) in PBS-BS-T for 1 h at 37°C. The pNPP enzymic substrate was added and the product was measured as described above. All incubations were followed by careful washing with PBS-T0.1%. All sera were simultaneously tested against the two antigens and their titration curves were compared.
6
Llama Serum Antibody Detection by ELISA
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Specific antibodies in individual serum samples were detected by indirect ELISA, using the antigen preparation described above, as coating antigen. Briefly, polystyrene microtiter plates (Nunc MaxiSorp, Rochester, New York) were coated with 1 μg/well of protein extract, during 1 h at 37°C. Afterwards, plates were washed with PBS containing 0.05% Tween 20 (PBS-T), and then blocked with 300 μl of 3% non-fat dry milk in PBS (PBS-M) overnight at 4°C. Plates were then washed three times with PBS-T and incubated with each llama serum, diluted 1:100 in PBS-M for 1 h at 37°C. After washing three times, bound antibodies were detected by incubating with 100 μl of a HRP-labeled goat anti-llama serum (Bethyl, Montgomery, USA) diluted at 1:7,000 in PBS-M, during 1 h at 37°C. Wells were washed, and the reaction was developed with 100 μl of the substrate-chromogen (3,3′,5,5′ tetramethylbenzydine) (Thermo Fisher Scientific, New York, USA). The reaction was stopped by the addition of 50 μl 4 N H2SO4. Optical density (O.D.) values at 450 nm were determined in a microplate reader (Original Multiskan Ex, Thermo Electron Corporation, Thermo Fisher Scientific, New York, USA). Given that there are no commercial nor in-house control sera available, data analysis was conducted as stated in the Section Statistical Analysis.
7
Quantifying Cytokine Secretion in Cell Cultures
8
Enzyme-Linked Immunosorbent Assay
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Polystyrene microtiter plates (Nunc, Roskilde, Denmark) were coated (overnight at 4 °C) with histone (2.5 μg ml−1), heparin sodium salt (2 μg ml−1), carbonic anhydrase (5 μg ml−1), actin or tubulin (5 μg ml−1), thyroglobulin (10 μg ml−1), F(ab′)2 fragments (1 μg ml−1) or trinitrophenyl–bovine serum albumin (10 μg ml−1) in 0.1 M carbonate-bicarbonate buffer, pH 9.6, whereas native DNA (10 μg ml−1) was coated in PBS, pH 6.8. The antigen-coated plates were washed with PBS, blocked with PBS–BSA, and then incubated with the samples (the three IVIg, Intraglobin F-IVIg affinity-purified antibody fractions and respective effluents; 150–1.17 μg ml−1) for 2 h at 37 °C. The wells were washed with PBS containing 0.1% Tween-20 (Merck, Darmstadt, Germany; PBS–T), and sheep anti-human IgG (H- and L-chain-specific) conjugated to horseradish peroxidase (0.8 μg ml−1 in PBS–T–BSA; Serotec, Oxford, UK) was added and incubated for 90 min at 37 °C. In human IgG F(ab′)2-coated plates, goat anti-human IgG (γ-chain-specific) conjugated to alkaline phosphatase (0.5 μg ml−1 in PBS–T–BSA; Sigma) was used. The enzyme substrate, tetramethyl-benzidine (Seramun, Germany) or p-nitrophenyl-phosphate (Sigma), was added and OD was measured at 450 or 405 nm, respectively, with an ExpertPlus microplate reader (Asys, Eugendorf, Austria).
9
PCV2 Antibody Detection ELISA
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Polystyrene microtiter plates (Nunc, Denmark) were coated with the purified recombinant Cap protein (50 ng per well) in 50 mM carbonate buffer (pH 9.6) overnight at 4°C. After absorption, the plates were washed three times with TBST and blocked with blocking buffer (5% skimmed milk powder in TBST) at 37°C for 30 min. Serum samples from the mice were diluted 1:50 in diluting buffer (diluted 1% skimmed milk powder in TBST) and incubated for 60 min at 7°C. After washing three times with TBST, plates were treated with HRP-conjugated goat anti-mouse immunoglobulin G (Sigma) diluted 1:8 000 in diluting buffer with 1% skimmed milk powder for 60 min at 37°C. The reaction was developed by using 3,3,5,5-tetramethylbenzidine (TMB) as the chromogen. The OD value of each well was measured at 450 nm using an ELISA reader. Serum samples with an optical density greater than those of normal mouse sera (means ± three standard deviations) were considered seropositive for PCV2.
10
Characterization of anti-proBDNF Antibody
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The purified monoclonal antibody against proBDNF was characterized by indirect ELISA27 (link) and Western Blot. For ELISA assay, 96-well polystyrene microtiter plates (Nunc, Roskilde, Denmark, USA) were coated with the generated human proBDNF prodomain, and commercial human, rat and mice proBDNF proteins (Alomone Labs, Israel), and human mBDNF (Alomone Labs, Israel) (all at 1 μg/mL with total volume 50 μL) overnight at 4 °C. The plates were washed three times in PBS–Tween-20 (0.05%, PBS-T), and the unbound sites were blocked with 100 μL of 2.5% skim milk at 37 °C for 1.5 h. The plates were washed as described above. The developed monoclonal anti-proBDNF antibody was added to the wells starting from 1:500 (37 °C for 1.5 h). After washing, HRP-conjugated rabbit anti-mouse immunoglobulin (Ig, 1:1000 v/v, Bio-Rad, Hercules, CA) was added and incubated for 1.5 h at 37 °C. The plates were washed, and the substrate solution containing tetramethylbenzidine (TMB) was added. The reaction was stopped by 20% H2SO4 after 15 min and the absorbance was measured at 450 nm by an ELISA reader (BioTek, U.S.).
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