RAW cells or BMDMs were seeded at a density of 1 × 106 cells per well of a 6-well tissue culture plate or at a density of 5 × 105 cells per well of a 12-well tissue culture plate in DMEM containing 10% neonatal calf serum 24 hours prior to stimulation. Media were replaced, and opsonized live KP serotype 2 (MOI 10:1, log phase) was introduced. KP was opsonized by incubating with 20% neonatal calf serum for 30 minutes at 4°C. Where indicated, leukoreduced sRBCs, hemin, CoPPIX, or PPIX were introduced concurrently. Leukoreduced sRBCs were resuspended in sterile PBS. In studies where hemin was added, hemin, CoPPIX, and PPIX were first dissolved in 100% DMSO at a concentration of 26 mg/mL (~40 mM) (8). Stock solution was then diluted in warmed DMEM to achieve a final concentration of 100 μM (final concentration of DMSO administered to cell = 0.25%). At the indicated time (usually 4 hours after KP infection), media were collected, spun at 10,000g for 10 minutes at 4°C, and cytokine release was evaluated in the resulting supernatant. Macrophages were washed with PBS and incubated in 800 μL to 1 mL of RBC lysis buffer (eBioscience, Invitrogen, 00-4333) for 30 to 60 seconds at room temperature with continuous swirling to lyse unengulfed RBCs. Macrophages were washed again with PBS and lysed to examine gene and protein expression via qRT-PCR and Western blot, respectively.

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