PMN were sorted using anti-Ly6C/Brilliant Violet 421 and anti-Ly6G/PerCpCy5.5 antibodies (Biolegend) and then incubated in RP-10(−) medium with PGE2 (final concentration 1 μM) or vehicle (ethanol) for 60 min at 37°C in 7% CO2. L. monocytogenes SD2710 (GFP+) was added and the plate was centrifuged for 5 min at 300 x g followed by incubation for 5 min at 37°C in 7% CO2. Polyclonal rabbit anti-Listeria O primary antisera (Difco) and goat anti-rabbit IgG–Texas Red secondary antibody (Thermo Fisher) were used to perform differential “in/out” staining as described previously (27 (link)). Cells were visualized using a Zeiss Axio Imager.Z1 with a 100x/1.4 NA PlanApo oil immersion objective and analyzed with AxioVision software. Slides were blinded and examined by two different investigators; average values are reported.
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Listeria Monocytogenes Infection Assay
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