Exosome-specific markers were quantified using a previously described dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) [55 (link), 56 (link), 64 (link)]. Samples were added to a high-binding 96-well microplate (DY990, R&D Systems). After overnight incubation at 4 °C, blocking with 1% BSA/PBS for 1 h at room temperature was performed. This was followed by primary antibody incubation at 1 μg/ml in PBS for 2 h at room temperature (CD9: Clone M-L13, BD Biosciences; CD63: Clone H5C6, BD Biosciences; CD81: Clone JS-81, BD Biosciences) and secondary antibody incubation at 0.25 µg/ml in PBS for 1 h at room temperature (biotin-conjugated goat anti-mouse IgG1, ab98691, Abcam). 1:1000 Eu-labelled streptavidin in DELFIA Assay Buffer (PerkinElmer) was then added and incubated for 1 h at room temperature. Finally, 100-µl DELFIA Enhancement Solution (PerkinElmer) was added to each well and time-resolved fluorimetry was performed using a PHERAstar plate reader (BMG Labtech) with excitation at 337 nm, detection at 620 nm, integration start at 60 µs, and integration time of 200 µs. Results are presented as arbitrary units (AU).
Ab98691
Manufactured by Abcam
Sourced in United States
Ab98691 is a lab equipment product. It serves as a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Dy990, by R&D Systems (3 mentions)
Pherastar plate reader, by BMG Labtech (3 mentions)
Delfia assay buffer, by PerkinElmer (2 mentions)
Delfia enhancement solution, by PerkinElmer (2 mentions)
Cd9 clone ml13, by BD (1 mentions)
Cd63 clone h5c6, by BD (1 mentions)
Cd81 clone js 81, by BD (1 mentions)
Tetramethylbenzidine, by Merck Group (1 mentions)
Streptavidin horseradish peroxidase hrp, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti cd9, by BD (1 mentions)
Clone js 81, by BD (1 mentions)
Clone n27f3 4, by Santa Cruz Biotechnology (1 mentions)
Clone h 300, by Santa Cruz Biotechnology (1 mentions)
4 protocols using ab98691
1
Quantification of Exosome-Specific Markers
2
Measuring Serum Anti-cBSA Antibodies
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Serum levels of anti-cBSA IgG (subclass IgG1 and IgG2a) were measured by ELISA. Briefly, Nunc-Immuno 96 MicroWell solid plates (Sigma-Aldrich Canada) were coated with 0.5 µg/well of cBSA (Chondrex, Inc.) in carbonate-bicarbonate coating buffer (50 mM Na2CO3, 50 mM NaHCO3, pH 9.6) at 4 °C overnight, and were blocked with 1% BSA (Sigma-Aldrich Canada) in PBS-T (PBS containing 0.05% Tween-20) buffer for 1 h at RT. After washing with PBS-T buffer, serum samples (100 µL/well) in triplicate, serially diluted with ELISA buffer (150 mM NaCl, 1 mM KH2PO4, 10 mm Na2HPO4, 2.6 mm KCl, 0.5% BSA, 0.1% Tween-20, pH 7.4), were incubated at 4 °C overnight. The serum anti-cBSA IgG1 and anti-cBSA IgG2a were detected by using biotinylated goat anti-mouse IgG1 (ab98691, Abcam, 1:1000 diluted) and anti-mouse IgG2 (ab98696, Abcam, diluted from 1:1000), respectively. The color was developed with streptavidin-horseradish peroxidase (HRP) and tetramethylbenzidine (Sigma-Aldrich Canada) and was stopped with 2 M H2SO4 solution. Similarly, serum complement C3 was determined by using mouse complement C3 ELISA kit (ab157711, Abcam) according to the manufacturer’s protocol. The serum titers of each substance were expressed as optical density (OD) at 450 nm (OD450). The “noise” associated with this protocol was confirmed to be low (approximately 5%) by three technical replicates.
3
Quantification of Exosome Markers by DELFIA
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Exosome markers were detected with DELFIA.34 Specifically, ExoDiff or ExoPr0 dilutions or their diluent, phosphate buffer saline (PBS), were loaded in duplicates on high‐binding 96‐well plates (R&D Systems, DY990) and incubated at 4°C. Next day, the plates were washed three times with DELFIA washing buffer before any reagent was added to wells (PerkinElmer, 1244‐114). First, plates were blocked with 1% BSA in PBS for 1 h at room temperature. Then, plates were incubated with anti‐CD9 (1:500), CD63 (1:200) or CD81 (1:500; BD Biosciences, 555 370, 556 019, 555 675). After 2 h, plates were incubated with appropriate secondary antibodies (1:2,000; Abcam, ab98691, ab97073) for 1 h. Afterwards, streptavidin‐europium conjugate in assay buffer (1:1,000) was added to wells for 1 h (PerkinElmer, 1244‐106 and 1244‐30). Finally, plates were washed six times and incubated with enhancement solution, while being shaken at 300rpm for 10 min (PerkinElmer, 1244‐104). The samples were analysed with a PHERAstar plate reader with the following settings: 337 nm excitation, 620 nm detection, 200 μs integration time and 60 μs lag time (BMG Labtech). Fluorescence value of each control, that is PBS incubated with both primary and secondary antibodies was subtracted from sample fluorescence.
4
Quantification of Extracellular Vesicles and Lipoproteins
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Specific markers of sEVs and lipoproteins were quantified using a previously described DELFIA [21 ,37 ] with modifications. Samples were added to a high-binding 96-well microplate (DY990, R&D Systems, Abingdon, UK). After overnight incubation at 4°C, blocking with 1% BSA/PBS for 1 h at room temperature was performed. This was followed by primary antibody incubation at 1 μg/ml in PBS for 2 h at room temperature (CD9: Clone M-L13, BD Biosciences, San Jose, USA; CD81: Clone JS-81, BD Biosciences; HSP70: Clone N27F3-4, Santa Cruz Biotechnology, Santa Cruz, USA; APOB: Clone H-300, Santa Cruz Biotechnology) and secondary antibody incubation at 0.25 µg/ml in PBS for 1 h at room temperature (biotin-conjugated goat anti-rabbit IgG for APOB, ab97073, Abcam or biotin-conjugated goat anti-mouse IgG1 for CD9, CD81 and HSP70, ab98691, Abcam). 1:1000 Eu-labelled streptavidin in DELFIA Assay Buffer (PerkinElmer, Beaconsfield, UK) was then added and incubated for 1 h at room temperature. Finally, 100 µl DELFIA Enhancement Solution (PerkinElmer) was added to each well and time-resolved fluorimetry was performed using a PHERAstar plate reader (BMG Labtech) with excitation at 337 nm, detection at 620 nm, integration start at 60 µs and integration time of 200 µs. Results are presented as arbitrary units (AU).
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