Bio plex cytokine assay kit

From a randomly selected subset of 20 longitudinal blood samples of the children aged 9 years, PT, FHA, Prn, and Td-specific enzyme-linked immunospot assays were performed on purified B-cells to determine the numbers of antigen-specific IgG producing memory B-cells (22 (link), 28 (link)). The same PBMC samples, depleted of CD19⁺ cells, were stimulated for 5 days with PT (heat inactivated), FHA, Prn, Td, or pokeweed mitogen and culture supernatants were stored at −80°C (23 (link)). In these supernatants, the cytokines interferon-gamma (IFN-γ) (Th1), interleukin-13 (IL-13) (Th2), IL-17 (Th17), and IL-10 (Treg) were quantified using the Bio-Plex cytokine assay kits (Bio-Rad Laboratories, Hercules, CA, USA) (27 (link)).

The spleen, thymus, and lymph nodes were weighed to evaluate responses of the immune system. To analyze cytokines related to the central and peripheral inflammatory responses, Th1 and Th2 cytokines from spinal cords and lymph nodes were measured using Bio-Plex cytokine assay kits (Bio-Rad, Richmond, CA, USA), according to the manufacturer's instructions. Briefly, anticytokine-conjugated beads were placed in 96-well microtiter plates and then removed by vacuum filtration. After then, samples, detection antibody, and streptavidin-PE were sequentially added and incubated for 30 min under mixing at 300 rpm. Final samples were immediately analyzed by a Bio-Plex array system, and cytokine concentrations were automatically calculated by Bio-Plex software (Bio-Rad).

At days 7, 14, and 21 days posttreatment the first bleomycin challenge, animals were anesthetized with isoflurane and sacrificed by bleeding from the abdominal aorta. Bronchoalveolar lavage fluid (BALF) was collected by gently washing the lungs with 0.6 mL sterile solution [Hank’s balanced salt solution × 10; ethylenediaminetetraacetic acid 100 mM; 4-(2-hydroxy-ethyl)-1-piperazineethansulphonic acid 1 mM; and distilled water] for three times in the bronchial tree and collected for subsequent analysis (14 (link)). After centrifugation at 400 g for 10 min, the BAL supernatants were frozen at −80°C for simultaneous quantitation of multiple cytokines/chemokines using a Bio-Plex™ Cytokine Assay Kit (Bio-Rad Laboratories, Segrate, Milano, Italy). The cell pellet was resuspended in 0.2 ml of PBS. Cell number was counted with an automated cell counter (Dasit XT 1800J). The concentration of MMP proteins in BAL fluid was determined by enzyme-linked immunosorbent assay (ELISA). MMP-2, MMP-9, and TIMP-1 were measured with specific ELISA kit (R&D System), and MMP-1 was measured with Mouse Matrix Metalloproteinase 1 ELISA Kit (MyBioSource) according to the manufacturer’s instructions.