Bio plex pro assay kit

Serum from aged mice was analyzed by custom Bio‐Plex Pro Assay Kit (Bio‐Rad) as per the manufacturer's instructions. Results were revealed using Bio‐Plex 200 System with the Bio‐Plex Manager 4 software.

Concentrations of murine Th1 cytokines interferon (IFN)-γ, Th2 cytokines IL-4, IL-5, IL-13 and RANTES/CCL5, pro-inflammatory cytokine IL-17 and anti-inflammatory cytokine IL-10 in serum and BAL fluid were determined using Multiplex MAP kit (EMD Millipore Corp., Billerica, MA, USA) and Bio-plex pro assay kit reagents using Luminex Bio-plex 200 suspension array system (Bio-Rad Corp. Hercules, CA, USA). The levels of IL-31 and IL-33 in BAL fluid of the mice were measured by enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific and BioLegend, San Diego, CA, USA, respectively). Th1/Th2 ratio was calculated based on the Th1 IFN-γ and Th2 IL-4 levels in BAL fluid. Murine OVA-specific IgE, total IgE, IL-9 and TGF-β concentrations in the serum and BAL fluid were determined using ELISA kits (BioLegend).

Whole brain lysates from mice treated with the three-injection regimen of LPS described above were gently homogenized in 150 μl of NP40 buffer (Invitrogen, Grand Island, NY). Samples were centrifuged for 5 min at 4 °C and 12,000×g. Protein content of the supernatant was then determined according to the BCA assay protocol supplied by the manufacturer. Protein concentrations are attached. A panel of 23 cytokines (interleukin (IL)-1α; IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-9; IL-10; IL-12(p40); IL-12(p70); IL-13; IL-17; eotaxin (CCL11); granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF); interferon (IFN)-γ; keratinocyte chemoattractant (KC) (CXCL1); monocyte chemoattractant protein (MCP)-1 (CCL2); macrophage inflammatory protein (MIP)-1α (CCL3); MIP-1β (CCL4); regulated on activation, normal T cell expressed and secreted (RANTES; CCL5) and tumor necrosis factor (TNF)-α) were measured in whole brain lysates using a murine Bio-Plex Pro™ assay kit (Bio-Rad Laboratories, Inc.; Hercules, CA). All samples were diluted 1:3 in sample diluent provided in the kit and processed according to the manufacturer’s protocol. Plates were read on a Bio-Plex 200 (Bio-Rad Laboratories, Inc.; Hercules, CA).

A panel of 23 cytokines (interleukin (IL)-1α; IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-9; IL-10; IL-12(p40); IL-12(p70); IL-13; IL-17; eotaxin (CCL11); granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF); interferon (IFN)-γ; keratinocyte chemoattractant (KC) (CXCL1); monocyte chemoattractant protein (MCP)-1 (CCL2); macrophage inflammatory protein (MIP)-1α (CCL3); MIP-1β (CCL4); regulated on activation, normal T cell expressed and secreted (RANTES; CCL5) and tumor necrosis factor (TNF)-α) were measured in serum and brain protein extracts using a murine Bio-Plex Pro assay kit (Bio-Rad Laboratories, Inc.; Hercules, CA, USA). Serum samples were diluted by adding 15ul serum to 100ul of sample diluent provided with the kit. Serum standards were diluted in the standard diluent provided with the kit. Brain samples were diluted by adding 40ul of protein extract to 100ul of sample diluent provided in the kit. Brain standard diluent was prepared by adding 2 parts brain homogenization buffer with 0.1% Triton X-100 and 1.5% bovine serum albumin (BSA) to 5 parts sample diluent provided with the kit. Plates were processed according to the kit procedures, and read on a Bio-Plex 200 (Bio-Rad Laboratories, Inc.; Hercules, CA).