Guava technologies

Cell cycle analysis was examined using propidium iodide staining to determine DNA content. The cells were trypsinized, washed twice with PBS and centrifuged at 500 × g for 5 min. Cells were then re-suspended in 0.5 mL PBS, and 10 mL 75% ethanol were added slowly while vortexing to prevent cell clumping. The cells were left in fixative at 4 °C for 30 min, centrifuged at 200 × g for 5 min and washed once with PBS supplemented with 1% bovine serum albumin (BSA). Cells were then suspended in fresh propidium iodide (Sigma, St. Louis, MO, USA)/RNase A (Qiagen) solution and incubated at 37 °C for 30 min. Samples were immediately analyzed by flow cytometry (Guava Technologies, Millipore, Billerica, MA, USA).

Female wild-type (WT) C57BL/6, NLRP3^-/-, and IL-18R^-/- mice aged 6–10 weeks were purchased from the Jackson Laboratory. ASC^-/- were a kind gift of Amy Hise (Case Western Reserve University, USA) and were bred in-house. All strains were on the C57BL/6 background and maintained in Specific Pathogen Free conditions. All animal experiments and protocols used in this study were approved by the Infectious Disease Research Institute’s Institutional Animal Care and Use Committee. Mice were immunized twice, 3 weeks apart via an intramuscular injection in the calf muscles of hind limb with 0.5 µg of ID93 or 0.1 µg rHA recombinant protein.^{48 (link)} Adjuvants were added as follows per dose: SE (2%), SLA (5 µg), Alhydrogel (100 µg aluminum) nanoalum (100 µg aluminum), and/or PAA (2.7 µg). For innate immune responses, draining inguinal lymph nodes were collected 6 h after immunization. For adaptive immune responses, spleens were collected in RPMI 7 days after the second immunization. Cells suspensions were obtained by manual disruption. Red blood cells contained in spleens were lysed using the Red Blood Cell Lysis Buffer (ThermoFisher). Total viable cells were counted (Guava Technologies, Millipore) and plated at 2 × 10⁶ cells/well in round-bottom 96-well plates.

The cells were pre-seeded on a 35-mm tissue culture dish (BD Pharmigen) at a density of 5000 cells/cm². Upon reaching 90% confluence, the cells were detached, fixed and permeabilized in 70% ethanol and left overnight at 4°C. Thereafter, 500 μl was extracted (containing 1 × 10⁶ cells), and DNA was stained with Propidium iodide/RNAse staining buffer (BD Pharmigen) for 15 min. at room temperature and subsequently washed in Dulbecco's PBS (DPBS; Invitrogen). DNA content was analysed on Guava Technologies (Millipore, Billerica, MA, USA) flow cytometer by using Cytosoft, Version 5.2, Guava Technologies software.