Measuring Ascorbate Levels in BMDMs

BMDMs were isolated and cultured for 7 days as described above. On day 7, cells were incubated with 50, 200 or 500 µM sodium ascorbate (Sigma Aldrich, St. Louis, MO, USA) and harvested at 30 min or 24 h to measure ascorbate content, or with 500 μM sodium ascorbate for 0, 2, 4, 6, 8 and 24 h. Wells were washed twice with PBS (Life Technologies, Carlsbad, CA, USA) and cells detached with two rounds of incubation in TrypLE (Life Technologies, Carlsbad, CA, USA) (200 µL each) and vigorous trituration. Cells were pelleted in microfuge tubes using a swing out rotor (500× g for 3 min at RT) and extracted with 0.54 M perchloric acid containing Diethylenetriamine penta-acetic acid (Sigma Aldrich, St. Louis, MO, USA) for ascorbate analysis [33 (link)]. Precipitated proteins were pelleted (10,000× g for 10 min at 4 °C) and ascorbate was measured in the supernatant using HPLC with electrochemical detection as previously described [34 (link)]. The concentration was assessed relative to standards ranging from 1.25 to 40 μM ascorbate made fresh for each analysis.