Ls6000 liquid scintillation analyzer

To measure oxidative PPP, the indicated cell types were plated onto 6 cm dishes, grown for 2d, washed twice with PBS, and the tissue culture media was incubated with media containing 0.8μCi of 1-¹⁴C-glucose (Perkin Elmer) for 5h. To capture gaseous ¹⁴CO₂, Whatman filter paper was taped to the inside of culture dish lid and the plate was then sealed with Parafilm; the experiment was terminated by adding 500μL of perchloric acid to the cells; the plate was resealed and kept at room temperature overnight. Filter papers were then carefully removed and placed into vials containing Scintisafe Plus 50% cocktail scintillation fluid (Fisher Scientific) and ¹⁴CO₂ activity was measured on a Beckman LS6000 Liquid Scintillation Analyzer. Notably, the incubation of cells with 6-¹⁴C-glucose, used to correct for 14CO₂ production from the citric acid cycle, yielded minimal ¹⁴CO₂ activity. To measure relative PPP via NMR, cells were maintained in DMEM containing 2-¹³C glucose for 18h. The next day, media was collected and ¹³C spectra acquired on above-mentioned 600 Mz INOVA spectrometer.

One million cells were plated onto 10 cm dishes in triplicate, grown overnight and treated with vehicle control (water) or with CQ or Q at the indicated doses for 3h at 37C. Cells were washed twice with PBS and the tissue culture media was replaced with media containing 0.8μCi of 1-¹⁴C-glucose (Perkin Elmer) or 6-¹⁴C-glucose containing vehicle (water) or the indicated antimalarial; cells were incubated for an additional 7h prior to analysis. To capture gaseous ¹⁴CO₂, Whatman filter paper was taped to the inside of culture dish lid and the plate was then sealed with Parafilm; the experiment was terminated by adding 500μL of perchloric acid to the cells; the plate was resealed and left in the incubator overnight. Filter papers were then carefully removed and placed into vials containing Ready-Safe Liquid Scintillation Fluid (Beckman Coulter), and ¹⁴CO₂ activity was measured on a Beckman LS6000 Liquid Scintillation Analyzer. The incubation of cells with 6-¹⁴C-glucose, used to correct for ¹⁴CO₂ production from the citric acid cycle, yielded minimal ¹⁴CO₂ activity.