|Figure 1. HEK-293T cells cultured in October 2016. 80% confluence after being seeded 1:25 four days prior. These cells were sent to transfection.|
Human embryonic kidney cells 293 (HEK-293) and 293T cells (those that contain SV40 Large T-antigen) show a reliable growth and have a propensity for transfection. Therefore, they are a major workhorse for research in cell biology. They are often used for expression because of quick replication rate. Especially important to this procedure is the replication rate which varies by the cell line that you are working with. HEK-293 cells see cell doubling once every 24–48 hours depending on the care. Most HEK-293T cells double every 12–20 hours depending on conditions. Our cell doubling time is typically closer to 12 hours. Establishing the rate at which your cells double is crucial for appropriate care and efficient use of your time in the lab. While this protocol is used for HEK-293T cells, it should be appropriate for any HEK cell line.
HEK cell behavior is important to understand in order to recognize the health of the cells and to appreciate the procedures that are used in the lab. Healthy HEK-293 cells grow on the surface of the cell culture plate. They adhere to the plate by proteins on the cell surface. Unhealthy cells do not have the same binding to the surface and thus float in the supernatant. Dead cells will appear much smaller than healthy cells with a black "ring" around them. Note that the cells in Figure 2, which are noticeably smaller and darker, are dead cells. One other quick way to monitor cell health is by the color of the media. DMEM with phenol red will begin to turn orange and yellow as more dead cells collect in the media, causing the media to become more basic. Note that a change in color could also indicate contamination. If there is a fogginess in your media, it is important to check whether the sample has been contaminated.
|Figure 2. HEK-293T cells cultured in October 2016. 70% confluence with dead cells after being seeded 1:10 three days prior. These cells were immediately split.|
Confluence of the cell culture is vital to the cell splitting process and is determined by how much of the plate is covered in cells. When checking your cells under the microscope, take note of how many cells are present. If there are only a few cells, then confluence is near 0%. However, if there is almost no "free" space on the plate then the culture is near 100% confluence. In Figure 1, cells are about 80% confluent while in Figure 2 they are around 60–70% confluent. Maintaining a good level of confluence is necessary for healthy cells. Cultures that are less than 5% confluent can struggle to grow while cultures near 100% confluence will have many dead cells.
NOTE: The steps that follow assume a 100 mm plate. When using other sized plates, scaling should be done according to surface area. Refer to Table 3 for appropriate scaling of common plate sizes.
|Type||Growth area (cm2)||Media required (mL)||Ratio to 100 mm dish|
|6 well plate||4.67||2.5||1:4|
|100 mm plate||55||10||1:1|
|100 cm2 flask||100||20||2:1|
|150 mm plate||152
|Figure 3. Common culture plate styles and scalings from this procedure.|