Human premixed multi analyte kit

Cytokine quantification was performed as described^{8 (link)}. All analytes were measured by magnetic luminex screening assay using Human Premixed Multi-Analyte Kit (R&D Systems) according to the manufacturers’ instructions. Measurements were performed with a Bioplex200 Analyzer equipped with Bio-Plex Manager^TM Software (Bio-Rad).

After treatment for 4 hours, concentrations of selected cytokines in the supernatants of ILC2 (approximately 6 × 10⁵ cells/well) cultures were measured with a Human Premixed Multi-Analyte Kit (R&D Systems, Minneapolis, Minn) with magnetic beads, according to the manufacturer's instruction. Results were obtained with a Bio-Plex 200 System (Bio-Rad Laboratories, Hercules, Calif).

Magnetic Luminex assays were performed according to the manufacturer's recommendations using a custom Human Premixed Multi‐Analyte Kit (R&D systems).

The specimens were thawed and they were allowed to slowly reach room temperature. Then, they were vortexed for 20 s. For milk degreasing, we centrifuged the tubes at 5000× g for 25 min at 4 °C. The skimmed milk was transferred into a new tube for further processing.
All breastmilk samples were checked for the presence of reactive anti-S1 RBD IgG antibodies using a sandwich enzyme-linked immunosorbent assay (ELISA), as previously described in Trofin et al. (2022) [25 (link)]. We used a 1:10 dilution in the sample diluent provided by the kit. The samples were processed in accordance to the manufacturer’s indications (TestLine Clinical Diagnostics, Czech Republic, Catalog number: CoRG96-EIA COVID-19 RBD IgG, test specificity of 99.15% and test sensitivity of 99.9%). The optical densities were measured with a TECAN Infinite 200 photometer at 450 nm wavelength, and the results were processed with the Magellan software.
Ten cytokines and chemokines were simultaneously measured from the same breastmilk sample using a bead-based immunoassay with the Luminex Multiplexing Assay. We followed the manufacturer’s instructions (Human Premixed Multi-Analyte Kit, R&D systems, Minneapolis, MN, USA, Catalog number: LXSHM-10). The samples were analyzed using a Luminex 200 (USA) device. We quantified the following cytokines: TNF-α, IL-6, IFN-β, IL-10, IL-1β, IFN-γ, IL-2, GM-CSF, IL-5 and IP-10.

The concentrations of the serum cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-9, and IL-17) were determined by xMAP multiplex technology [17 (link)] on the Luminex 100 system (Luminex, Austin, TX, USA), using a Human Premixed Multi-Analyte Kit (R&D Systems, Inc., Minneapolis, USA), and further analyzed using Bioplex Manager Software (Bio-Rad Life Sciences, CA, USA). All analyses were carried out in compliance with the manufacturers' instructions. Standard curves were generated for each cytokine, and the mean fluorescence intensity (MFI) of each cytokine in each well was converted into a concentration using the linear portion of the standard.