Growing Human Cells in Culture
Transfer and Viability
Now that you have your mammalian cells growing well in culture (if they are not contaminated), you need to transfer the culture again and check the viability. As you have found by doing your eyeball check of your cultures, there are some floating cells that are probably dead. What about the cells that are attached? Are there many that have also lost viability? You can check this with Trypan Blue.
As you pass the cells to new culture flasks, you will save 100 ul of cells for the viability assay. This sample will be mixed with Trypan Blue. Trypan Blue is actively transported out of the cells. By using a hemocytometer and a microscope, you can figure out the following:
1. Go through the same pass procedure that you did last week.
2. When you get to the point of the transfering cells to the two new flasks, aseptically pipet out a 100 ul cell sample into a new separate tube.
3. Add 0.1 ml of the Trypan Blue dye to the cells in the sample.
4. Add a drop of the Trypan Blue/Cell sample to a hemocytometer and cover slip set up.
5. Under low power and low light, count the total number of
cells and the number of stained cells in the five big squares
(as shown below). We will then figure out the number of cells,
viable cells, and percent viability.