Extraction of aqueous metabolites
from the cells and tissue samples for NMR experiments was carried
out as described earlier.30 (link) MDA-MB-231
cells were cultured and treated with indicated concentrations of DIM
for 24 h. After treatment, cells were scraped off from the flask,
collected into a tube, and washed with cold 1×PBS to remove media
traces. Further, 600 μL of chilled methanol was added to the
cells and mixed using a roto-spin rotary mixer overnight at 4 °C.
Later, 600 μL of distilled water and chloroform were added to
the cell suspension and then vortexed for 30 s and centrifuged at
150×g for 5 min. The upper aqueous fraction
was collected into a new tube and dried by a vacuum evaporator (Eppendorf,
Hamburg, Germany). Before analysis, the extracts were reconstituted
in 55 μL of PBS prepared in D2O (100 mM concentration)
and 495 μL of D2O.
from the cells and tissue samples for NMR experiments was carried
out as described earlier.30 (link) MDA-MB-231
cells were cultured and treated with indicated concentrations of DIM
for 24 h. After treatment, cells were scraped off from the flask,
collected into a tube, and washed with cold 1×PBS to remove media
traces. Further, 600 μL of chilled methanol was added to the
cells and mixed using a roto-spin rotary mixer overnight at 4 °C.
Later, 600 μL of distilled water and chloroform were added to
the cell suspension and then vortexed for 30 s and centrifuged at
150×g for 5 min. The upper aqueous fraction
was collected into a new tube and dried by a vacuum evaporator (Eppendorf,
Hamburg, Germany). Before analysis, the extracts were reconstituted
in 55 μL of PBS prepared in D2O (100 mM concentration)
and 495 μL of D2O.
