Tissue culture treated plates

Murine RAW 264.7 macrophages from ATCC were grown in DMEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% nonessential amino acids, and 1% sodium pyruvate. Cells were grown to 80% confluency in 5% CO₂ in 96-well tissue culture treated plates (Greiner Bio-One) at 37°C. DMEM was then replaced with RPMI medium lacking phenol red with 5% fetal bovine serum, 1% L-glutamine, 1% nonessential amino acids, and 1% sodium pyruvate two hours prior to start of the assay. RB50 or RB50ΔclpV were added to plates at an MOI of 100 and the plates were centrifuged at 5000 RPM for 5 min at RT and were incubated in 5% CO₂ at 37°C. One hour later, the supernatant was replaced with 100 μg/mL gentamicin in RPMI lacking phenol red to kill extracellular bacteria and the plates were incubated once again in 5% CO₂ at 37°C. After one hour or 24 hours, supernatants were collected from wells and lactate dehydrogenase (LDH) release was measured to quantify levels of cytotoxicity using a Cytotox96 kit (Promega) according to the manufacturer’s instructions.