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The beads were analyzed using the Bio-Plex System (Bio-Rad Laboratories, HercUSAs, USA).
Beads were analyzed using a Beckman Coulter EPICS ALTRA flow cytometer (High Wycombe, Bucks, UK) and data analyzed with Bender MedSystems software.
Movements of the fluorescent beads were analyzed using the automatic spots-tracking module of the Imaris software package (Bitplane, Inc). to generate a set of position lists.
SIRT2 was detected by Western blot analysis using anti-His6 antibody, and the GST fusion proteins attached on the Glutathione-Sepharose beads were analyzed using ponceau S staining.
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Bead velocities were analyzed using the ImageJ manual tracking plugin.
Tracked bead displacements were analyzed using the two-tailed Student's t test for p-values and are reported as mean ± SEM.
500 ng from each of the 14 RNA pools were hybridised simultaneously to 16 arrays (including 2 arrays for the technical replica experiment) contained in 2 RatRef-12 Expression BeadChips, as per Illumina protocol (Illumina, San Diego, USA). the Bead-chip output files were analyzed using the BeadStudio-3 San Diego(IllUSAnas San Diego, USA) as previously reported [ 6].
Supernatants were initially analyzed by ELISA and cytometric bead array kits (CBA); however, to gain a more global analysis of multiple cytokine/chemokine production, supernatants were analyzed using Milliplex bead assays.
Concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α in CSF and serum (only in patients with bacterial meningitis) were analyzed using a cytometric bead array kit (BD™ Cytometric Bead Array – Human Inflammatory cytokine kit) and with a three-colour flow cytometer FACSCalibur™ (both BD Biosciences, San Jose, CA, USA).
At least 50 events per bead were read and the data were analyzed using Millipex Analyst software (EMD Millipore).
Levels of IL-6 and IL-8 in PBMC supernatants were analyzed using single-bead kits (LHC0061 and LHC0081, Invitrogen), whereas a six-plex kit was used (with an extracellular protein buffer reagent kit [LHB0001], Invitrogen) for detection of IL-1β (LHC0011), IL-4 (LHC0041), IL-10 (LHC0101), IFN-γ (LHC4031), GM-CSF (LHC2011) and TNF (LHC3011, all from Invitrogen) in the cell supernatants.
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