Milliplex map human cytokine kit

2 x 10⁴ DCs were cultured in 96-well flat-bottom plates with 100 μL of complete RPMI medium containing 10 % human AB serum (HS) for 2 days in the absence or presence of CD40 ligand-transfected cells (CD40L Tx, 5 x 10⁴ cells/well). CD40L Tx was kindly provided by Prof Giovanna Lombardi.⁵⁷ (link) IL-10 levels in the supernatant were assayed by using the MILLIPLEX MAP Human Cytokine Kit (Millipore) and acquired on a Luminex 100 flow-based sorting and detection analyzer (Luminex Corporation).

6 ml of whole blood samples were collected with ethylenediaminetetraacetic acid (EDTA) as anticoagulant before intake of the next dose of methadone. The plasma was obtained from the supernatant of whole blood after centrifugation at 2000×g in a Kubota 2800 centrifuge (Kubota Co., Osaka, Japan) for 20 min at 4°C, then dispensed into 1 ml/microcentrifuge tube and frozen at -80°C until use.
Cytokine IL-7 level was determined using the Milliplex^® MAP human cytokine kit (Millipore, Billerica, MA). All sample data were acquired from a MAGPIX^® System (Luminex Corp., Austin, TX).

Cytokines were quantified in AM supernatants using a Milliplex MAP Human Cytokine Kit (Millipore, #HCYTOMAG-60K) as detailed in Hoppstädter et al. (23 (link)). AMs were kept at a density of 1 × 10⁵ cells per well in 96 well plates in a total volume of 100 μL medium in the presence or absence of Pam₃CSK₄ (100 ng/mL) or Poly(I:C) (10 μg/mL) for 6 h. The supernatants were collected and stored at −80°C until they were used in the multiplex cytokine assay. The immunoassay procedure was performed using a serial dilution of the 10,000 pg/mL human cytokine standard according to the manufacturer's instructions, and the plate was read at the Luminex 200 System (Millipore).
Murine TNF-α was quantified by bioassay as previously described (11 (link)). L929 cells were seeded at a density of 3 × 10⁴ cells per well into a 96-well plate. After 24 h, the medium was replaced by 100 μL of actinomycin D solution (1 μg/mL in standard medium) and cells were incubated for 1 h at 37°C. Subsequently, BMM supernatants (100 μL per well) were added. Dilution series of recombinant murine TNF-α (100–2,500 pg/mL) were run alongside the samples to generate a standard curve. The plates were incubated for an additional 24 h at 37°C. The MTT assay (see Determination of Cell Viability) was used to assess cell viability.

The concentrations of 26 cytokines [including IL-1β, IL-2, IL-6, IL-7, IL-12p70, GM-CSF, IFN-γ, TNF-α, IL-1α, IL-8, IL-12p40, IL-15, eotaxin, fractalkine 3, G-CSF, MCP-1, MIP-1α, and−1β, RANTES, ITAC, monokine-induced by interferon Gamma (MIG), MIP-3α and TGF-β1, β2 and β3] were measured in semen using a human cytokine milliplex MAP kit (Millipore Corporation, St. Charles, Missouri, USA), according to the manufacturer's protocol. Seminal plasma samples were thawed and filtered by centrifugation using 0.2 μm cellulose acetate filters (Sigma, USA) prior to cytokine/chemokine measurements, to exclude debris (3 (link)). All values below the detection limit were recorded as half of the lowest measured concentration for each cytokine.