Acetyl-CoA measurement

Acetyl-CoA generated by in vitro cultured Mcu^fl/fl or Mcu^Δmye BMMs with or without L. monocytogenes challenge were quantified using the Acetyl-CoA Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, 50 μL of fresh sample was prepared for each reaction (well), then, 50 μL of the appropriate reaction mix was added to each of the wells. Mixed well and incubated the reaction for 10 minutes at 37 °C. The fluorescence intensity (E_x:E_m= 535:587 nm) was measured. The mount of Acetyl-CoA in sample well was calculated using the following equation:

The concentration of acetyl-CoA in the test samples was calculated as: concentration of acetyl-CoA = Ay/Sv; Ay = amount of acetyl-CoA in sample well (pmol); Sv = sample volume (μL) added into the reaction well. Each sample was assayed with triplicate.