Facsdiva software version 8

Single-cell suspensions were centrifuged at 450 × g for 7 min, resuspended in ice-cold staining buffer (1× PBS with 2% FBS), and placed in 96-well round-bottom plates. After incubation with purified anti-FcγRII/III antibody (clone 2.4G2) at 4 °C for 15 min, cells were stained with a mixture of fluorescent surface antibodies at 4 °C for 30 min. The stained samples were either directly analyzed by flow cytometry or fixed with 2% formalin at 4 °C for 20 min prior to storage at 4 °C overnight. The full list of antibodies used can be found in Supplementary Table 1. Flow cytometry was performed with FACSAria™ II or FACSCelesta™ flow cytometer (BD Biosciences). Data were acquired using BD FACSDiva software version 8.0.2 (BD Biosciences) and analyzed using FlowJo 10.5.3 (BD Biosciences).
The CD45⁺CD11c^hiSiglec-F^hi lung AMs of adult Hdac3^fl/fl and Ubc^CreERHdac3^fl/fl mice were sorted by FACS Aria II flow cytometer. The CD45⁺F4/80^hiCD11b^int fetal lung macrophages at E14.5, CD45⁺F4/80^intCD11b^intpreAMs at E16.5 and E18.5, CD45⁺CD11c^hiSiglec-F^hi adult lung AMs, as well as CD45⁺F4/80^intCD11b^hi FL monocytes at E16.5 from Hdac3^fl/fl and Csf1r^iCreHdac3^fl/fl mice were also sorted by FACSAria™ II flow cytometer. The purity of the isolated populations was >95%.

The cell cultures were dissociated using Dispase (1 mg/mL) (Stemcell Technologies, Vancouver, BC, Canada) and then labeled with SIRPa-PE antibodies (#323806, Biolegend, San Diego, CA, USA) at the recommended concentrations. The stained cells were analyzed using the FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA) with BD FACSDiva^TM software, version 8.0 (BD Biosciences, San Jose, CA, USA).

For fluorescence–based flow cytometry, bacterial cells were washed two times with fresh mineral salt medium in which the substrates were omitted and diluted 1:100 in tethering buffer (10 mM K₂HPO₄, 10 mM KH₂PO₄, 10 mM lactic acid, 0.1 mM EDTA, and 1 μM L-methionine, pH 7.0). Fluorescence-activated cell analysis was carried out on a BD LSRFortessa^TM SORP flow-cytometer (BD Biosciences, Franklin Lakes, NJ, USA; Piatkevich and Verkhusha, 2011 (link); Telford et al., 2012 (link)). Fluorescence was detected using a 561-nm laser (yellow–green) at 100 mW for excitation and a 610/20 bandpass filter. The forward and side scatter values were monitored using a 488-nm laser (blue) at 50 mW. The acquired data were analyzed using BD FACSDiva^TM software version 8.0 (BD Biosciences, Franklin Lakes, NJ, USA) with data collected in FCS 3.0 file format.

Surface antigen expression was detected by flow cytometry. Cells were resuspended in ice-cold PBS with 2 mM EDTA and stained for 30 min at 4 °C using 5 µL of two-color antibody solutions αCD45-FITC/αCD14-PE, αCD3-FITC/αCD19-PE, αCD3-FITC/αHLA- DR-PE, or αIgG1-FITC/αIgG2a-PE from the BD Simultest IMK Plus reagent kit, single-color antibodies αCD23-PE or αIgG1-PE control (all from BD Biosciences, Franklin Lakes, NJ, USA). After washing with 1 mL PBS/EDTA solution, cells were analyzed on a LSRII cytometer using FACSDiva software version 8.0.1 (BD Biosciences, Franklin Lakes, NJ, USA).

Blood was collected into Lithium-heparin tubes. An antibody mastermix containing anti-CD3 (SP34- 2, AF700; BD Pharmingen), anti-CD4 (L200, APC-H7; BD Pharmingen), anti-CD8 (SK1, PerCP; Biolegend) and anti-CD69 (FN50, PE-Cy7; eBioscience) was added to 100 µL of blood. Samples were incubated for 30 min at 4 °C, and washed three times in PBS by centrifugation at 314 × g for 5 min. The cells were resuspended in 400 µL of PBS, and immediately analysed using the LSR II flow cytometer (BD Biosciences), FACSDiva software version 8.0.1 (BD Biosciences), and FlowJo software version 10 (FlowJo, LLC). The gating strategy in Supplementary Fig. S2 was used to identify activated T cells.