I will measure the internalization of proteins using pHrodo in a time-dependent manner. However, i have limited protein. Therefore, I would like to measure the track the same cells at 5-6 time points: 1, 3, 8, 12, 24, and 48 hours. I will keep the lid on the plate, and the measurements will be short, conducted at 37°C with the plate reader next to the incubator. I'm wondering if repeatedly measuring the fluorescence could affect the signal over time. Is it feasible to conduct these measurements without significantly impacting the cells or the accuracy of the signal, especially with the repeated exposure to fluorescent light?
I don't think that a taking a couple of images will cause significant photobleaching, so I don't think this will be a problem. What you have to make sure is that the indicator stays in the cells for 2 days. I would say that there will be gradual loss of the indicator. Since you want to measure pH and assume that the fluorescence intensity is proportional to pH, this will result in errors. I would suggest using a ratiometric pH probe, such as BCECF. Even though this indicator may also be lost from the cells, it is the ratio of two fluorescence intensities that will tell you the pH and the ratio will not be affected by indicator loss.
It would make sense to look at the cells under a microscope to see the distribution of the indicator in cells, and also to check how cell viability is affected by the indicator being present for an extended period of time.
Another control would be to load the indicator into normally cultured cells without any treatment and see if you get roughly the same pH for these untreated cells for 48 hours.
Best of luck,
Peter
Dear Peter Nagy,
Thank you for your answer.
I am a bit confused with BCECF suggestion. I will label the proteins I want to follow with pHrodo to see internalization to endolysosomal system. As far as I understand BCECF is measuring cytosolic pH.
I assume fluoresence will reach a peak after a time-point and after that it will reduce which means it will be delivered to endolysosomes and after that either pHrodo will be degraded or will be transferred to cytosol or outside cell.
I misunderstood your question, I thought you are using this indicator:
https://www.thermofisher.com/order/catalog/product/P35373
but actually you must be using this:
https://www.thermofisher.com/order/catalog/product/P35355
And your aim is not to measure pH, but to track the labeled proteins intracellularly. I don't think that repeated exposure of cells to excitation light according to the plan you mentioned will affect the results.
You obviously have to consider that if your protein escapes from the endolysosomal system, phrodo fluorescence will significantly decrease:
https://www.thermofisher.com/hu/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-70/phrodo-ph-sensors.html
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