Bio plex workstation

To evaluate the effects of Ly6G-versus Gr-1-mediated cell depletion on
the inflammatory milieu during S. aureus orthopedic biofilm
infection, a custom-designed mouse microbead array was used (MILLIPLEX;
Millipore Corporation, Billerica, MA), which detects the following mediators:
G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5,
IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, CCL2, CCL3, CCL5, CXCL1,
CXCL2, CXCL9, CXCL10, TNF-α and VEGF. A Bio-Plex workstation (Bio-Rad,
Hercules, CA) was used to analyze results and values were normalized to the
total amount of protein recovered from each sample.

Mice were euthanized at the indicated intervals after infection and perfused transcardially with ice-cold PBS. Brain abscesses were dissected within 1–2 mm of the lesion margins and immediately disrupted in 500 μl of homogenization buffer [1X PBS supplemented with a protease inhibitor cocktail tablet (Roche, Mannheim, Germany) and RNase inhibitor (Promega, Madison, WI)]. Serial 10-fold dilutions of brain abscess homogenates were plated on trypticase soy-agar (TSA) plates supplemented with 5% sheep blood (Hemostat Laboratories, Dixon CA) to determine bacterial titers (expressed as log₁₀ CFU/g wet tissue weight). To compare proinflammatory mediator expression profiles between KO versus WT mice, a multi-analyte microbead array was utilized according to the manufacturer’s instructions (MILLIPLEX; Millipore, Billerica, MA) that detects IL-1α, IL-1β, TNF-α, IFN-γ, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-15, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CCL2, CCL3, CCL4 and CCL5. Results were analyzed using a Bio-Plex workstation (Bio-Rad, Hercules, CA) and adjusted based on the amount of total protein extracted from brain abscess homogenates for normalization using a colorimetric Bio-Rad DC Protein Assay kit (Bio-Rad). IL-1β was also measured by sandwich ELISA (BD OptIEA, San Diego, CA).