Milliplex map human cytokine chemokine multiplex immunoassay

hTMSCs were plated at a density of 1 × 10⁵ in 24-well plates, allowed to adhere overnight, and pre-treated with TLR3 or TLR4 agonists for 1 hr as previously described [12 (link)]. IL (interleukin)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-α, GM-CSF, and IFN-γ were analyzed with the Milliplex Map human cytokine/chemokine multiplex immunoassay (Millipore, Billerica, MA). These experiments were conducted at least three times on each hTMSCs pools.

hTMSCs were plated at a density of 1×10⁵ in 24-well plates, allowed to adhere overnight, then pre-treated with TLR agonists (LPS for TLR4 or poly(I:C) for TLR3) for 1 hr as indicated. Conditioned medium was collected after 48 hr and analyzed with MILLIPLEX MAP human cytokine/chemokine multiplex immunoassay (Millipore, Billerica, MA) for IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10 (CXCL10), RANTES (CCL5), TNF-a, GM-CSF, and IFN-γ following the manufacturer's instructions. These experiments were performed at least three times on three individual MSC donor pools.

hNTSCs were seeded at a density of 2 × 10⁴ cells in 24-well plates and allowed to adhere overnight. Conditioned media acquired from hypoxic and normoxic control conditions on days 0, 7, and 14 were stored at −80 °C. Cytokines assays were performed on all five cell lines and average values were calculated. The medium samples were analyzed using the MILLIPLEX MAP human cytokine/chemokine multiplex immunoassay (Millipore, Billerica, MA, USA) to detect various interleukins (IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12), IP-10 (CXCL10), RANTES (CCL5), tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-γ in accordance with the manufacturer’s instructions. These experiments were conducted independently on at least three occasions using individual MSCs donor pools.