How to know when your cells are confluent and ready to be subcultured?

As a researcher or cell culturist, one of the most important skills you need to master is understanding when your cells are ready to be subcultured. In this blog post, I, Nic, will be explaining the various methods you can use to determine when your cells have reached confluency and are ready to be passaged to a new dish.

First, let's define what we mean by "confluent cells." Confluent cells are cells that have grown and spread to the point where they have completely covered the surface of the dish or flask in which they are growing. These cells are not just growing in clumps, but have formed a single, unified layer.

There are a few different methods you can use to determine when your cells have reached confluency. One of the most common methods is to visually inspect your cells under a microscope. At low magnification, you should be able to see that the cells have spread to cover the entire surface of the dish or flask. However, at high magnification, you may be able to see that there are still small areas of the dish or flask that are not covered by cells, indicating that the cells are not yet confluent.

Another way to determine confluency is to use a microscope and measure the percentage of the area that is covered by cells. Using gridlines or software that overlays a grid onto your microscope's live feed. By counting the number of grid squares that are covered by cells and comparing it to the total number of grid squares, you can calculate the percentage of confluency.

A more practical method is to measure the size of the culture, for example by counting the number of cells or using a hemocytometer. With experience, you can estimate the confluency of your culture by the cell number.

Another method of determining confluency is to use a dye that stains cells that are in contact with each other. These dyes will only stain cells that are touching one another, which means that if all of the cells are touching, the entire culture will be stained. This method can be useful because it allows you to quickly and easily determine if your cells are confluent, even if you are unable to see them under the microscope.

Finally, it's important to keep in mind that the rate of cell growth and confluency is often dependent on the cell type and culture conditions. With experience, you'll get to know how long it takes for your specific cells to become confluent under the conditions you are culturing in. Additionally, certain media or supplement may also affect the rate of cell growth and confluency.

In conclusion, determining when your cells are confluent and ready to be subcultured is crucial for maintaining healthy and productive cell cultures. By visual inspection, measuring the percentage of the area covered by cells, counting the number of cells, staining the cells, or having experience with the specific cell type and culture conditions, you will be able to determine when your cells are confluent and ready to be passaged to a new dish.

As always it is important to establish and stick to a protocol that works well for your cell type, and also document it properly. This will help ensure that your cell cultures are consistently healthy and productive.

Great, now that we know what confluent cells are and some of the ways we can check if our cells are confluent, let's make it easy for you to follow. Here is a step-by-step guide to checking if your cells are confluent and ready to be subcultured:

Step 1: Look at your cells under a microscope. You can use low or high magnification, but make sure you can see the entire dish or flask.

Step 2: Check to see if the cells have spread out to cover the entire surface of the dish or flask. If there are still small areas without cells, then your cells are not yet confluent.

Step 3: Count the number of cells in your culture. You can use a hemocytometer or try to estimate the number of cells by eye. With experience, you'll get to know how many cells you should expect for a confluent culture.

Step 4: Use a special dye that only stains cells that are touching each other. If all the cells are touching, the entire culture will be stained. This is a quick way to see if your cells are confluent.

Step 5: Compare the number of cells or the percentage of confluency with the previous time you check or the expected numbers for your cell type and culture conditions.

Step 6: If your cells are confluent, it's time to subculture them! This means taking some cells out of the dish or flask they're growing in and putting them in a new dish or flask with fresh growth medium. This way your cells will continue to grow and be healthy.

Here's a checklist for you to make sure you're following all the steps correctly:

•  Look at cells under microscope
•  Check if cells have spread to cover the entire surface
•  Count or estimate the number of cells
•  Use special dye to check confluency
•  Compare with previous time or expected numbers
•  Subculture cells if confluent

By following these steps and checking off this checklist, you will be able to tell if your cells are confluent and ready to be subcultured. Remember, it's important to establish and stick to a protocol that works well for your cell type and also to document it properly. This will help ensure that your cell cultures are consistently healthy and productive.