Multi screen 96 well plates

We carried out a multispacer sequence typing (MST) technique on all fecal specimens positive by PCR-sequencing as previously described in our laboratory [23 (link),45 (link)]. PCRs were realized in a 2720 Thermal Cycler (Applied Biosystems, Foster City, California, USA) and followed all the steps described for standard PCR used for the molecular analysis of fecal specimens. Negative controls consisting of PCR mixture without DNA template were included in each PCR run. All PCR products were sequenced in both directions using the same primers as used for PCRs in a 2720 Thermal Cycler (Applied Biosystems) with an initial 1-min denaturation step at 96 °C, followed by 25 cycles denaturation for 10 s each at 96 °C, a 20 s annealing step at 50 °C, and a 4-min extension step at 60 °C. Sequencing products were purified using the MultiScreen 96-well plates Millipore (Merck, Molsheim, France), containing 5% of Sephadex G-50 (Sigma-Aldrich), and sequences were analyzed on an ABI PRISM 31309 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) and edited using the ChromasPro software (version 1.42; Technelysium Pty Ltd., Tewantin, Australia). For each intergenic spacer, a spacer type (ST) was defined as a sequence exhibiting unique genetic polymorphism (SNPs and indels). MST genotypes were defined as a unique combination of the four spacer sequences [23 (link),45 (link)].

Multiscreen 96-well plates (Millipore) were coated with 50 μl sterile PBS (Tritium) containing either 5 μg/ml MenC-polysaccharide (NIBSC) conjugated to human serum albumin (NIBSC), 5 μg/ml tetanus toxoid (Baxter), 10 μg/ml goat-anti-human IgG (Cappel) or 10 μg/ml goat-anti-human IgA (Cappel) or solely PBS (blank wells). Coated plates were stored at 4°C until use.
After 5 days of polyclonal stimulation, B cells were harvested, washed, resuspended in AIMV medium (Gibco, Invitrogen) and counted. Wells were blocked with 100 μl AIMV medium for 1 hour before use and filled with 50 μl of cell suspension containing 1x10⁵ B cells. The plates were incubated ON at 37°C and 5% CO₂. The next day, wells were washed with PBS/0.05% Tween20 (Merck) and goat-anti-human IgG (Sigma Aldrich, 1/5000) or goat anti-human IgA (Sigma 1/2500) was added to bind to secreted IgG or IgA, respectively. After 1 hour of incubation at 37°C, the wells were washed and bound conjugate revealed by adding a substrate (5-bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium, Sigma). Spots were counted using an automatic ELISpot reader (C.T.L. Immunoscan). The number of MenC-PS-specific and TT-specific memory B cells per sample was measured in triplo and the mean number of these three measurements was used for the statistical analyses.

Cytokines and chemokines secreted into the cell culture supernatant from microglia were assayed with a multiplex rat cytokine chemokine assay kit (Millipore Sigma, St. Louis, MO, USA), following the manufacturer’s instructions. Five cytokines, including proinflammatory and anti-inflammatory cytokines (interleukin (IL-1β, IL-4, IL-6, IL-10 and tumor necrosis factor (TNF-α), and MCP-1 were analyzed. Results were compared across groups in which equal numbers of cells were evaluated. The assays were performed in triplicate using Millipore multi-screen 96-well plates. Data were collected using MagPix (Luminex, Austin, TX, USA). Data analysis was performed using the Millipore immunoassay curve fitting software Belysa 1.1 for Luminex XMAP ™. A five-parameter logistic formula was used to calculate the sample concentrations from the standard curves.

Splenocytes, lung, and bone marrow cells were isolated from tissue samples at 5 d.p.c.. RSV F-specific antibody secreting cells were determined on Multi-screen 96 well plates (Millipore, Billerica, MA) pre-coated with purified recombinant F protein (400ng/ml) by enzyme-linked immunospot (ELISpot) assay as previously described [42 (link)]. The spots of anti-F secreting cells were developed with biotinylated anti-mouse IgG and streptavidin-AP (Southern) and 3,3’-diaminobenzidine tetrahydrochloride (DAB, Thermo Scientific). The spots of IFN-γ and IL-4 secreting cells were determined on Multi-screen 96 well plates coated with cytokine specific capture antibodies as described [40 (link)–42 (link)]. Briefly, lung cells (0.2x10⁶) per well were cultured with peptide F_92-106 (ELQLLMQSTPATNNR, 4 μg/ml), F_85-93 (KYKNAVTEL), G_183-195 (WAICKRIPNKKPG), and M2_82-90 (SYIGSINNI) as previously described [43 (link), 44 (link)]. After 36 h stimulation, the spots were developed with biotinylated mouse IFN-γ or IL-4 antibody, alkaline phosphatase labeled streptavidin (BD Pharmingen) and then counted using an ELISpot reader (BioSys, Miami, FL).