Percp conjugated anti mouse cd45

In a separate study, ears of mice were intradermally injected with C. acnes alone, C. acnes/β-ionone or C. acnes/luteolin, as described above. Twelve hours later, the ears were excised (n = 5), split into dorsal and ventral halves, and then dispersed by passage through a 70 μm cell strainer (BD Biosciences) into the RPMI medium. Each sample was then brought to a final volume of 4 mL with RPMI medium. Cells were washed with PBS and filtered again through a 40 μm cell strainer (BD Biosciences). Single-cell suspensions were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ly6G, a neutrophil marker (BioLegend, San Diego, CA, USA), and peridinin chlorophyll protein (PerCP)-conjugated anti-mouse CD45, a common leukocyte marker (BioLegend), in fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5% w/v bovine serum albumin and 0.09% sodium azide) for 30 min and washed three times with FACS buffer. For intracellular cytokine analysis, cells were washed, surface stained as described above, fixed and permeabilized for intracellular staining of allophycocyanin (APC)-conjugated anti-IL-1β (eBioscience, San Diego, CA, USA), as instructed by the manufacturer. Samples were analyzed with FacsCantoII (BD Biosciences) using FACS Diva software, and the data were analyzed using FlowJo software.

To analyze the leukocyte population in the blood, 100 μL aliquots of whole blood were incubated with antibodies against mouse leukocyte surface antigens at 4 °C in the dark for 30 min. The antibodies included peridinin-chlorophyll (PerCP)-conjugated anti-mouse CD45 (Biolegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ly6G (Biolegend), Pacific blue-conjugated anti-mouse F4/80 (Biolegend), phycoerythrin (PE)-conjugated anti-mouse Ly6C (eBioscience, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-mouse C–C chemokine receptor type 2 (CCR2) (R&D Systems, Minneapolis, MN, USA), and Alexa Fluor^® 700-conjugated anti-mouse C-X3-C motif chemokine receptor 1 (CX3CR1) (Biolegend).
After red blood cells were lysed, stained cells were suspended in staining buffer (PBS with 0.5% bovine serum albumin) and analyzed with an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA, USA). CD45-positive (CD45⁺) cells were gated and determined to be leukocytes. Leukocyte populations are presented as percentages of neutrophils (Ly6G⁺), macrophages (F4/80⁺), inflammatory monocytes (Ly6C^higCCR2⁺), and anti-inflammatory monocytes (Ly6C^lowCX3CR1⁺) among leukocytes (CD45⁺).