We have now performed experiments in HT29 and HepG2 cells to test mitigation/synthetic lethality between…
Plate 8000 cells/well (2.5*10^4/cm2) of each cell line in 70 uL/well media in a 96 well format (2 x 48 wells per cell line—4 replicates each x 12 doses x 2 drugs). Make a mastermix for 48*1.1 wells and aliquot to wells using multichannel pipettor to be as even as possible.
Add 10 uL/well of a mix of OptiMEM + drug (prepared in 96-well round bottom plate to use multi-channel pipettor
Monitor cells the next day (24 hrs after plating and drug addition) and make notes of cell death trends. Do not change media.
The next day (48 hrs after plating and drug addition), perform flow cytometric analysis on 2 of the 4 wells/cell line and drug condition (protocol below). Feed the remaining 2 of 4 wells/cell line and drug condition with the same drug dose using 190 uL media volume per well + 10 uL OptiMEM + drug—increase drug amount proportionally.
Perform flow cytometric analysis 4-5 days post-initial drug addition on this second set of wells.
Flow cytometric analysis
There is no need to perform flow cytometry on all doses. Determine doses that are reasonably close to the dose with ~50% cell death. Include 2-3 concentrations above and below that concentration as well as the DMSO/water control (6-8 concentrations total). Use 2 of the 4 replicate wells per round of flow cytometry. Aspirate media. Add 50 uL PBS/well. Collect PBS. Trypsinize cells using multichannel pipettor with 30 uL/well trypsin. Collect trypsin. Quench with 20 uL/well media. Collect. Before FACS, add 20 uL media + appropriate propidium iodide (PI) concentration. Transfer to FACS tubes if unable to use 96-well automated flow cytometry and perform flow cytometry, noting cell concentration as well as dead cells (since often dead cells float away). Make sure to use same flow rate for each sample so that you control for cells/second through the machine.
The 50% cytotoxic concentration (CC50) will be extrapolated from the plot of drug concentration versus live (PI-) cells/second (we usually gate a segment of the time axis from 10-30 seconds after starting collection)
by using regression analysis. Drug concentration will be plotted to X axis. Live cell number (a) or ratio (b) will be plotted to y axis.