Figure 9.

Washing cells following MTT incubation removes intracellularly formed extracellular formazan. Light microscopy images (×4) showing the effect of washing the cells’ supernatant before solubilizing formazan on removing formazan crystals. 20,000 PC-3 cells were seeded per well of 96-well plates and were allowed to grow in phenol red-free RPMI with FCS for 26 h. The cells were then incubated with 0.4 mg/mL MTT for 3 h. The wells were then divided into 2 groups: (a) Non-washed wells, (b) Washed wells in which the supernatant was totally removed, and then cells were washed two times with PBS and fresh media (with the same volume as non-washed wells) was added to each well. Red arrows show the extracellular needle-shaped formazan crystals which are much fewer than the non-washed sample.