24 well glass bottomedcell culture plates

A431 and CAF cells were removed from the cell culture dishes with
trypsin and re-suspended in sterile 0.25% methylcellulose solution in DMEM. The
cellulose solution contained a 1:1 ratio of A431 and CAF cells at a
concentration of 1×10⁶ cells per ml. 20 μl droplets
were plated onto the underside of a 10 cm culture dish and allowed to form
spheroids in a 37 °C incubator overnight. The spheroids were then
embedded in a collagen I/matrigel gel mix at a concentration of approximately 4
mg/ml collagen I and 2 mg/ml matrigel (BD Bioscience) in 24-well glass-bottomed
cell culture plates (MatTek) on a 37C hot block. The gel was incubated for at
least 30 min at 37°C with 5% CO_2. The gel was covered with
DMEM media containing 10 % FCS. 60 hours later, the spheroids embedded in the
gel were washed with PBS then fixed for 20 min at room temperature with 4%
paraformaldehyde. The spheroids were then imaged with an inverted Zeiss LSM780
at a magnification of 10×, 20× and 63×. 100-150 μm
of z stack images were collected and image stacks were processed by ZEN software
(Carl Zeiss) to yield maximum-intensity projections.