Incucyte live cell analysis imaging system

Cells were trypsinized, counted, plated in 2 mL complete RPMI at 10,000 cells per well of a 12-well plate, and allowed to attach overnight. Cells were then washed two times with 1 mL PBS per well, then treated with 2.5 mL of the indicated medium. A replicate plate was counted to determine starting cell number at the time of treatment. 72 hr post-treatment, cells were counted to calculate proliferation rate. Cell counts were determined using a Multisizer 3 Coulter Counter (Beckman Coulter) with a diameter setting of 10–30 μm. Proliferation rate was determined using the following formula:
$$\text{Proliferation}\text{rate}\left(\text{Doublings}\text{per}\text{day}\right)=\left[{\log }_2\left(\text{Final}\text{cell}\text{count}/\text{Initial}\text{cell}\text{count}\right)\right]/3\text{days}$$
For assessment of proliferation by continuous live cell imaging, cells were trypsinized, pelleted and washed with PBS, counted, resuspended in the appropriate medium, then plated directly into clear 96-well plates at the cell densities as indicated. Plates were placed into an IncuCyte Live Cell Analysis Imaging System (Sartorius), and images were acquired every 3 hr using the 10x objective. Cell confluence was determined from a mask generated by the IncuCyte Zoom Analysis S3 2017 software’s standard settings.