Serum were isolated and stored at -20°C for batch testing for selected soluble mediators using human magnetic bead Luminex multi-analyte assay following the manufacturer’s protocol (Bio-techne custom analyte mix). Results were obtained with a Bio-Plex 200 System (Bio-Rad). CSF-1 was measured by standardized sandwich ELISA (R&D Systems).
Luminex multi analyte assay
Manufactured by Bio-Techne
The Luminex multi-analyte assay is a platform for the simultaneous detection and quantification of multiple analytes in a single sample. It utilizes color-coded microbeads to capture specific target analytes, which are then detected and measured using a specialized Luminex instrument.
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3 protocols using luminex multi analyte assay
1
Serum Soluble Mediator Profiling
2
Viral Decontamination and Cytokine Analysis of SARS-CoV-2 Samples
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Plasma samples were put on ice and collected as soon as possible after arrival in the lab. Before protein analysis, plasma and nasal samples were treated in a P3 laboratory for viral decontamination using a protocol previously described for SARS-CoV, which we validated for SARS-CoV-2 [14 (link)]. Briefly, samples were treated with 1% TRITON X100 (vol/vol) and 0.3% tri-N-butyl phosphate (vol/vol) for 2 h at room temperature. Tri-N-butyl phosphate was removed before cytokine analysis by passing the treated samples though C18 columns. IL-6, TNF and IL-10 were measured with a commercial triplex assay (Quanterix) on a Simoa HD-1 analyzer (Quanterix). An additional 38 cytokines and chemokines were measured in plasma supernatants with a commercial Luminex multi-analyte assay (Biotechne, R&D systems). Data were acquired on a Bio-Plex 200 System (Bio-Rad) and analyzed with Bio-Plex Manager v5 (Bio-Rad).
3
Cytokine Quantification in Inactivated Nasopharyngeal Samples
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Nasopharyngeal samples were inactivated with Triton X-100 1% (v/v) for 2h at RT, and cytokines were quantified as described [29] . IFNα2, IFNγ, IFNλ3, IL-17A were quantified with Simoa assays developed with Quanterix homebrew kits as described [30] . Other cytokines and chemokines were measured with a commercial Luminex multi-analyte assay (Biotechne, R&D system).
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