To characterize leukocyte infiltrates in inflamed soft tissues
surrounding the knee joint during S. aureus biofilm infection,
tissues were excised, dissociated using the rubber end of a plunger from a 3 cc
syringe, and passed through a 35 µm filter (BD Falcon, Bedford, MA). The
resulting filtrate was washed with 1× PBS and cells were collected by
centrifugation (300 × g, 10 min), whereupon RBCs were
lysed using BD Pharm Lyse (BD Biosciences; San Diego, CA). After lysis, cells
were resuspended in PBS containing 2% FBS, followed by incubation in Fc
Block (BD Biosciences, San Diego, CA) to minimize non-specific antibody binding.
Cells were stained with CD45-APC, Ly6G-PE, Ly6C-PerCPCy5.5, F4/80-PE Cy7,
CCR2-FITC (R&D Systems; Minneapolis, MN), and CD11b-eFluor450. All
fluorochrome-conjugated antibodies were purchased from BD Biosciences (San
Diego, CA) or eBioscience (San Diego, CA) unless otherwise indicated. An aliquot
of cells was stained with isotype-matched control antibodies to assess the
degree of non-specific staining and fluorescence minus one was used to identify
gating thresholds (22 (link)). The number of
events analyzed ranged from 20,000–100,000 per sample, depending on the
experimental setup. Analysis was performed using BD FACSDiva software with cells
gated on the total leukocyte population (CD45+).
surrounding the knee joint during S. aureus biofilm infection,
tissues were excised, dissociated using the rubber end of a plunger from a 3 cc
syringe, and passed through a 35 µm filter (BD Falcon, Bedford, MA). The
resulting filtrate was washed with 1× PBS and cells were collected by
centrifugation (300 × g, 10 min), whereupon RBCs were
lysed using BD Pharm Lyse (BD Biosciences; San Diego, CA). After lysis, cells
were resuspended in PBS containing 2% FBS, followed by incubation in Fc
Block (BD Biosciences, San Diego, CA) to minimize non-specific antibody binding.
Cells were stained with CD45-APC, Ly6G-PE, Ly6C-PerCPCy5.5, F4/80-PE Cy7,
CCR2-FITC (R&D Systems; Minneapolis, MN), and CD11b-eFluor450. All
fluorochrome-conjugated antibodies were purchased from BD Biosciences (San
Diego, CA) or eBioscience (San Diego, CA) unless otherwise indicated. An aliquot
of cells was stained with isotype-matched control antibodies to assess the
degree of non-specific staining and fluorescence minus one was used to identify
gating thresholds (22 (link)). The number of
events analyzed ranged from 20,000–100,000 per sample, depending on the
experimental setup. Analysis was performed using BD FACSDiva software with cells
gated on the total leukocyte population (CD45+).