Alexa fluor 488 anti cd11b

Fresh bone marrow cells were used to generate BMDMs, using L929-cell conditioned
medium (LCCM) as a source of macrophage colony stimulating factor [23 (link)]. Cells were resuspended in 10 ml bone marrow
differentiation media (R20/30; composition: RPMI1640 supplemented with 20% fetal
bovine serum, 30% LCCM, 100 U/ml penicillin, 100 mg/ml streptomycin and 1 mM
sodium pyruvate) and were seeded in untreated 90×15 mm culture dishes and
incubated at 37°C in a 5% CO₂ atmosphere. Three days after
seeding, an extra 10 ml of fresh R20/30 were added per dish and incubated for additional 3
days. On day 6, the attached cells were washed and detached with 10 ml of cold sterile
PBS. Cells were centrifuged at 200 × g for 5 minutes and resuspended in 10 ml of
BMDM cultivation media (R10/5; composition: RPMI 1640, 10% fetal bovine serum,
5% LCCM). Cells were seeded at a density of 10⁶/ml on the day before
the experiments. The macrophage purity of these preparations was usually
>95% as assessed by flow cytometry using murine macrophage antibody Alexa
Fluor 488 anti-CD11b and APC anti- F4/80 (Biolegend). For infection with
P.gingivalis, a multiplicity of infection (MOI) of 100 was used for all
the experiments as described in the figure legends.

Twenty-four hours after receiving saline or P. aeruginosa, mice were euthanized and bronchoalveolar lavage (BAL) was obtained by repeated instillation/withdrawal of ice cold 1mL PBS containing 5mM EDTA and 0.5% BSA (total volume collected 4-5mL per mouse). BAL samples were stained with the following antibodies to identify neutrophils: APC/Cy7 anti-Ly6G, Alexa Fluor 488 anti-CD11b, PE anti-CXCR2 (all from BioLegend). Samples were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi) and post-acquisition analysis was performed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ). Group sizes for analyses of BAL neutrophils were: Hem-Saline: n = 4; Sham-PA, Hem-PA: n = 7.