Human high sensitivity t cell magnetic bead panel kit

Serum Dp- and Df-specific IgE levels were measured by fluorescent enzyme immunoassay using the ImmunoCAP^® system (Thermo Fisher Scientific, Uppsala, Sweden). IL-4, IL-5, IL-10, IL-13, and IFN-γ levels were measured in serum using a Human High Sensitivity T-Cell Magnetic Bead Panel kit (Millipore, Billerica, MA, USA) and Luminex LX200 (Millipore). Urinary LTE₄ and PGF_2α levels were measured using a commercial ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) and expressed as ng/mmol creatinine. Creatinine level was measured using a CREJ2 kit with Cobas 8000 (Roche Diagnostics, Mannheim, Germany).

For measuring the levels of the cytokines
IL-6, TNFα, IL-1β, IL-8, IL-10, IL-12, and IFN-γ,
cell culture supernatants were collected from the human macrophage
chambers after co-culture and transfection. The supernatants were
stored at −80 °C and thawed to RT. They were centrifuged
at 1500g for 10 min to exclude any cell debris during
the collection process. A commercially available, Milliplex, human
high-sensitivity T-cell magnetic bead panel kit (Millipore, USA) was
used to measure the levels of the above cytokines in the supernatants,
following the manufacturer’s instructions. The fluorescence
intensity was detected using the MAGPIX system (Luminex, USA). The
standard kit and cell samples were added in duplicate wells. Cytokine
concentrations were calculated after the collection of standard curves
and used at the protein level (pg/mL). The data were processed using
Milliplex analyst software (version 5.1 Flex, VigeneTech, USA). Control
supernatants were the ones obtained after macrophage stimulation with
small-GO-PEG-PAMAM alone with co-culture and without transfection.