Quantifying Hypoxia in Cell Spheroids

FaDu spheroids were grown using the liquid overlay technique, and HCT116 and H1299 spheroids were grown in lipidure-coated U-bottom plates (UMS Bio). Half of the medium was replaced twice per week. Spheroid hypoxia was quantified by EF5 staining, using an anti-EF5 antibody (both reagents obtained from University of Pennsylvania)7 (link). Two hundred micromolar EF5 was added to the spheroids for 6 h before fixation in 4% paraformaldehyde for 24 h, incubation in 30% sucrose for 2.5 h, and then addition of OCT (VWR, TissueTek) for cryosectioning. Spheroid sections were incubated for 40 min in TNB blocking reagent (Perkin Elmer), washed for 5 min in 1 × PBS, 0.3% Tween 20, and then incubated overnight with 70 μl of undiluted 75 μg ml⁻¹ anti-EF5 antibody. Three washes of 45 min were then performed using 1 × PBS, 0.3% Tween 20 before addition of DAPI vectorshield mounting medium (Vector Laboratories) and fluorescence microscopy (Nikon 90i, Nikon). Spheroid diameter was derived from the spheroid cross-sectional area measured by the Gelcount colony counter (Oxford Optronix) or Axiovert200M (Carl Zeiss MicroImaging GmbH).