In my previous blog I mentioned that I would be conducting a cell assay this week. I was able to do two of them, however the second assay had some technical difficulties.
On Monday, I checked on the cells that had begun growing last week and saw that there was a sufficient cell/mL density for an assay. I did two assays per cell well, one with MTT dye and another using crystal violet. The well was split horizontally so that each assay would have an equal number of cells and controls. The assay takes anywhere between 4-8 hours, depending on how quickly the cells crystallize. This assay was done using breast cancer cells, which are larger, so the crystallization period was only an hour, rather than four. The rest of the time comes from mixing the cells. Once dyed, the cells have to be placed on a cell mixer for 1-4 hours, depending on how quickly the crystals are broken down. They should be checked every half an hour under the microscope. Once the assay is finished, the cell well is put through a cell reader to determine the OD number per well.
Planning on doing another assay, I began growing cells on Tuesday. This time, I grew colon cells at densities of 4,000, 8,000, 10,000, 12,000, 15,000, 20,000 and 25,000 cells/mL. I used another cell well but grew the cells horizontally, rather than by columns. I also graphed by data from the first assay using excel.
On Thursday the cells were ready and I repeated the assay from Monday, using rrystal Violet and MTT again. However, I forgot to split the plate vertically. On Monday, the plate was split horizontally because the cells were grown in columns. On Tuesday, I had grown the cells in rows to better utilize the plate but forgot to switch that when doing the dyeing. This way, the crystal violet assay was done using densities 4,000-12,000 while the MTT assay was done with densities 15,000-25,000 and no control. I will redo the assay next week.