Hello Krish Dodani

If the media was exhausted, there would be a shift in pH and thus in color, right?

Yes.

If there was not meant to be any shift in color with these cells, I do not see why the company would sell a version of this media with phenol red in it.

Phenol red is a pH indicator. It is used in culture media to indicate when the media requires changing or if there is some possible bacterial contamination in the media. Generally, the culture media must be changed after every second day even if the color of the culture media remains red. You may put the cells at risk if you wait for the media to turn yellow.

Although they do not change color, I very often observe round, refractive particles that look really similar to cells when I add trypsin to them which leads me to believe these particles are unattached cells. I am guessing these unattached cells are dead cells.

Sometimes, because of stressful condition (which may arise due to various reasons in culture), the cells may fail to attach or may even die. So, you may be right that those round, refractive particles may represent dead cells or debris. Try to identify the source of stress in culture.

You may check for the following.

1. Check for the presence of contamination.

2. Check whether there is temperature fluctuation in the incubator.

3. Check for insufficient or inappropriate cell culture surface.

4. Check whether there is an inappropriate environmental gas mixture.

5. Check whether the cells are provided with sufficient nutrients in the culture media.

6. Do not overexpose cells to trypsin. Generally, 3-4mins trypsin exposure should suffice.

The fluctuation that you observe may not be normal. Try considering the above suggestions.

Hope they help!

Best.