Cell-free DNA was quantified in sera and plasma using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) according to the manufacturer’s instructions. MPO-DNA complexes were quantified similarly to what has been previously described (33 ). This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human MPO antibody (Bio-Rad, 0400-0002), diluted to a concentration of 5 μg/ml in coating buffer (Cell Death kit). The plate was washed 3 times with wash buffer (0.05% Tween 20 in PBS), and then blocked with 1% bovine serum albumin in PBS for 90 minutes at room temperature. The plate was again washed 3 times, before incubating overnight at 4°C with 10% sera or plasma in the aforementioned blocking buffer. The plate was washed 5 times, and then incubated for 90 minutes at room temperature with 1x anti-DNA antibody (HRP-conjugated; Cell Death kit) diluted in blocking buffer. After 5 more washes, the plate was developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Invitrogen) followed by a 2N sulfuric acid stop solution. Absorbance was measured at a wavelength of 405 nm with a Synergy HT Multi-Mode Microplate Reader (BioTek). Data was normalized to an in vitro-prepared NET standard, included on every plate.
High binding eia ria 96 well plate
Manufactured by Corning
The High-binding EIA/RIA 96-well plate is a laboratory equipment product designed for enzyme-linked immunosorbent assay (EIA) and radioimmunoassay (RIA) applications. The plate features a high-binding surface that optimizes the capture and detection of target analytes in these assay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cell death detection elisa kit, by Roche (8 mentions)
Anti human mpo antibody, by Bio-Rad (5 mentions)
Synergy ht multi mode microplate reader, by Agilent Technologies (4 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (3 mentions)
Tetramethylbenzidine tmb substrate, by Thermo Fisher Scientific (2 mentions)
Anti human myeloperoxidase antibody, by Bio-Rad (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
Biotin anti mouse ige, by BD (1 mentions)
Anti mouse igg hrp, by Southern Biotech (1 mentions)
Anti mouse ige, by BD (1 mentions)
Anti mouse igm hrp, by Southern Biotech (1 mentions)
Anti mouse iga hrp, by Southern Biotech (1 mentions)
Streptavidin hrp, by Rockland Immunochemicals (1 mentions)
Synergy 2 plate reader, by Agilent Technologies (1 mentions)
10 protocols using high binding eia ria 96 well plate
1
Quantification of Cell-free DNA and MPO-DNA Complexes
2
Quantification of MPO-DNA Complexes
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MPO-DNA complexes were quantified similarly as previously described (59 (link)). This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human MPO antibody (Bio-Rad0400-0002) and diluted to a concentration of 0.5 μg/mL in coating buffer (Cell Death Detection ELISA kit). The plate was washed 3 times with wash buffer (0.05% Tween 20 in PBS) and then blocked with 1% BSA in PBS for 1 hour at room temperature. The plate was again washed 3 times, before incubating for 1 hour at room temperature with 1:500 mouse serum in the aforementioned blocking buffer. The plate was washed 5 times and then incubated for 1 hour at room temperature with 1× anti-DNA antibody (HRP-conjugated; Cell Death Detection ELISA kit) diluted 1:100 in blocking buffer. After 5 more washes, the plate was developed with 3,3′,5,5′-TMB substrate (Invitrogen), followed by a 2 N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm with a Synergy HT Multi-Mode Microplate Reader (BioTek). Data were normalized to an in vitro–prepared NET standard included on every plate.
3
Quantification of MPO-DNA Complexes
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MPO-DNA complexes were quantified similarly to what has been previously described (86 (link)). This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human MPO antibody (Bio-Rad 0400-0002), diluted to a concentration of 1 μg/ml in coating buffer (Cell Death kit). The plate was washed two times with wash buffer [0.05% Tween 20 in phosphate-buffered saline (PBS)], and then blocked with 4% bovine serum albumin in PBS (supplemented with 0.05% Tween 20) for 2 hours at room temperature. The plate was again washed five times, before incubating for 90 min at room temperature with 10% serum or plasma in the aforementioned blocking buffer (without Tween 20). The plate was washed five times and then incubated for 90 min at room temperature with 10× anti-DNA antibody [horseradish peroxidase (HRP) conjugated; from the Cell Death kit] diluted 1:100 in blocking buffer. After five more washes, the plate was developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Invitrogen) followed by a 2N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Data were normalized to in vitro–prepared NET standards included on every plate, which were quantified on the basis of their DNA content.
4
Quantifying Myeloperoxidase-DNA Complexes in Sera
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Myeloperoxidase-DNA complexes were quantified in sera similarly to what has been previously described [27 (link), 28 ]. This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4ºC with anti-human myeloperoxidase antibody (Bio-Rad 0400-0002), diluted to a concentration of 1 µg/ml in coating buffer (Cell Death kit). The plate was washed two times with wash buffer (0.05% Tween 20 in PBS), and then blocked with 4% bovine serum albumin in PBS (supplemented with 0.05% Tween 20) for 2 h at room temperature. The plate was again washed five times, before incubating for 90 min at room temperature with 10% serum in the aforementioned blocking buffer (without Tween 20). The plate was washed five times, and then incubated for 90 min at room temperature with 10 × anti-DNA antibody (HRP-conjugated; from the Cell Death kit) diluted 1:100 in blocking buffer. After five more washes, the plate was developed with 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Invitrogen) followed by a 2 N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Data were normalized to in vitro-prepared NET standards included on every plate.
5
Quantification of NET Formation
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This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human neutrophil elastase antibody (MilliporeSigma, 481001), diluted to a concentration of 5 μg/mL in coating buffer (Cell Death kit). Next, the plate was washed 3 times with wash buffer (0.05% Tween 20 in PBS) and then blocked with 1% BSA in PBS for 1 hour at room temperature. Next, the plate was washed 3 times before incubating overnight at 4°C with 10% human plasma in the blocking buffer. Next, the plate was washed 3 times and then incubated for 1 hour at room temperature with 1× anti-DNA antibody (HRP conjugated; Cell Death kit) diluted 1:100 in blocking buffer. After 5 more washes, the plate was developed with 3,3′,5,5′-TMB substrate followed by a 2N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm with a Synergy HT Multi-Mode Microplate Reader. Data were normalized to an in vitro–prepared NET standard and included on every plate.
6
Quantification of MPO-DNA Complexes
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MPO-DNA complexes were quantified similarly to what has been previously described (38 (link)). This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4°C with anti-human MPO antibody (Bio-Rad 0400-0002), diluted to a concentration of 0.5 μg/mL in coating buffer (Cell Death kit). Next, the plate was washed 3 times with wash buffer (0.05% Tween 20 in PBS) and then blocked with 1% BSA in PBS for 1 hour at room temperature. Next, the plate was washed 3 times before incubating for 1 hour at room temperature with 1:500 mouse serum in the blocking buffer. Next, the plate was washed 5 times and then incubated for 1 hour at room temperature with 1× anti-DNA antibody (HRP conjugated; Cell Death kit) diluted 1:100 in blocking buffer. After 5 more washes, the plate was developed with 3,3′,5,5′-TMB substrate followed by a 2N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm with a Synergy HT Multi-Mode Microplate Reader (BioTek). Data were normalized to an in vitro–prepared NET standard and included on every plate.
7
Quantification of Myeloperoxidase-DNA Complexes
8
Quantification of MPO-DNA Complexes
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MPO-DNA complexes were quantified similarly to what has been previously described60 (link). This protocol used several reagents from the Cell Death Detection ELISA kit (Roche). First, a high-binding EIA/RIA 96-well plate (Costar) was coated overnight at 4 °C with anti-human MPO antibody (Bio-Rad 0400-0002), diluted to a concentration of 2 µg ml−1 in coating buffer (Cell Death kit). The plate was washed three times with wash buffer (0.05% Tween 20 in PBS), and then blocked with 1% bovine serum albumin in PBS for 90 min at room temperature. The plate was again washed three times, before incubating for 1 h at room temperature with 10% serum or plasma in the aforementioned blocking buffer. The plate was washed five times, and then incubated for 90 min at room temperature with 1x anti-DNA antibody (HRP-conjugated; Cell Death kit) diluted 1:20 in blocking buffer. After five more washes, the plate was developed with 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate (Invitrogen) followed by a 2 N sulfuric acid stop solution. Absorbance was measured at a wavelength of 450 nm with a Synergy HT Multi-Mode Microplate Reader (BioTek). Data were normalized to an in vitro-prepared NET standard, included on every plate.
9
Enzyme Immunoassay for Antibody Detection
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High-binding EIA/RIA 96-well plates (Costar) were coated overnight with 10µg/mL BSA-NP32 or BSA-NP5 (Biosearch Technologies Inc) diluted in ELISA coating buffer (28.6mM Na2Co3, 11.9mM NaHCO3, pH 9.6), or 2µg/mL anti-mouse IgE (BD 553413) diluted in PBS. Wells were subsequently washed four times with 0.05% Tween20/PBS, then blocked with 3% BSA/PBS for 2 hours at room temperature. After washing, sera was serially diluted in 1% BSA/PBS, added to wells and incubated for 2 hours at room temperature. Wells were washed and incubated with anti-mouse IgG-HRP (1030-05), anti-mouse IgM-HRP (1021-05), anti-mouse IgA-HRP (1040-05) from Southern Biotech, or anti-mouse IgE-biotin (BD 553419) diluted in 1% BSA/PBS for 2 hours at room temperature. Biotinylated antibodies were further incubated with streptavidin-HRP (Rockland) for 40 minutes at room temperature. After washing, HRP was detected with 1X TMB ELISA Substrate Solution (eBioscience) and color development was stopped with 1M orthophosphoric acid. Plates were analyzed at 450nm with a Biotrak II plate reader.
10
Cell Attachment Assay Protocol
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