12-Well Tissue Culture

A 12-well tissue culture plate was used to carry out the LIVE/DEAD Assay. Well “A” contained the untreated HepG2 cells. Well “B” contained camptothecin, which is used as a control for killing the HepG2 cells instead of the use of plumbagin. Well “C” contained the compound Epigallocatechin Gallate. Well “D” remained empty, because a vehicle treatment is not needed for this experiment. Each well contained 1 ml of HepG2 cells. A negative and positive control where both set up in the 12-well tissue culture plate. The positive control was set to be Well “B”, where the HepG2 cells where treated with camptothecin. The negative control was Well “A”, that had the HepG2 wells only. The cells where left for 24 hours to allow for adhesiveness. After time …show more content…
For camptothecin, 50µl of stock was added. The 12-well plate was then incubated for an additional 24 hours. The 12-well plate was removed from the incubator, and placed in a flume hood. With the use of a P1000 pipette, and carefully making sure not to starch the bottom of the well with the pipette; all the media within the well was removed and disposed in the Waste container. This step was done to each well, with changing the pipette tip each time after disposing the waste. 1 mL of PBS was added per well, once added to all the wells; the PBS was removed and disposed in the waste container as well. 250µl of the LIVE/DEAD staining solution was added to each well. The LIVE/DEAD staining solution contained (10 mls of sterile PBS, 20µl of 2mM EthD-1 stock solution, 5µl of 4mM calcein AM stock solution). The 12-well plate was covered to allow for 20 mins of incubation at room temperature. After the 20 minutes the remaining steps where preformed fast, so the photobleaching would not occur with the LIVE/DEAD staining solution. 1ml of PBS was added to the wells again, once all wells received the PBS; the PBS and LIVE/DEAD staining solution is removed per well and disposed in the waste