Dulbecco s pbs with calcium and magnesium

At least 80,000–100,000 purified AMs or IMs (see below) were plated in µ-Slide 8 Well chamber slides (Ibidi) and cultured in macrophage culture medium (DMEM/F12 media supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, and 50 ng/ml M-CSF), and incubated at 37°C and 5% CO₂ for 1–3 days to allow cells to rest. Infections of macrophages were performed in supplemented DMEM with 2% FBS at MOIs of 0.05 or 0.1, which was chosen to minimize widespread cell death and potential intercellular effects of infection. After 2 h, cells were gently washed with Dulbecco’s PBS with calcium and magnesium (Gibco), followed by replacement with macrophage culture medium.

For in vitro experiments, 1.5 × 10⁶ bone marrow-derived dendritic cells (BMDCs) were plated into 12-well suspension plates in BMDC growth media. Cells were pulsed for 2 h at a concentration of 1.4 mg/ml with C1498-OVA AMCNPs, equivalent C1498-OVA WCL vaccine, or equivalent CpG plus 1 μg/ml OVA SIINFEKL peptide (InvivoGen, San Diego, CA), then washed twice with fresh media. After 48 h of additional culture, cells were then collected in PBS with 1 mM EDTA and washed twice in PBS with 1% bovine serum albumin (Thermo Fisher Scientific). Cells were stained as indicated (Supplementary Table 1). For in vivo experiments, 50 µL of 25 mg/ml of AMCNPs, equivalent WCL vaccine, or mock treatment, were inoculated subcutaneously into each hock [36 (link)] of 8–12-week-old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME). After 24 h, the popliteal lymph nodes of the mice were collected and manually dissociated in 500 µl dissociation buffer [Dulbecco’s PBS with calcium and magnesium (Gibco, Waltham, MA), 1 mg/ml collagenase D (Roche, Basel, Switzerland), and 1 mg/ml DNase I grade II (Roche)]. Cells were stained as indicated (Supplementary Table 1).