Did you continue with your culture? Did your cells look healthy? If it is contamination by something your cells most likely will show poor growth, the number of the structures may increase, the culture media will exhaust quickly. Can you add antibiotics to you medium to reduce the possibility of contamination? Another possibility: check whether these structures/contaminants are already in your media by imaging you medium alone.

Good luck!

Hello Kamilla Batista da Silva Souza

It cannot be indicative of contamination because contamination inside cells is generally not observed as bright areas under phase contrast microscopy. Instead, contaminants like bacteria often appear as darker, distinct structures or as an overall increase in the background darkness. This is because phase contrast microscopy utilizes differences in the refractive index between the cell and the surrounding medium, which are then translated into contrast changes in the image.

These small and bright structures could be vesicles because under the microscope they can be visualized as bright spots due to the contrast they create against the surrounding cytoplasm. Moreover, the movement of vesicles is often random, due to factors like Brownian motion and the activity of molecular motors.

If you do consider other organelles such as the mitochondria, it is not possible because mitochondria, along with other organelles, appear dark against a lighter background. This is because thicker organelles like mitochondria have a higher refractive index, causing them to appear darker due to the interference of light waves.

Best,

THP-1 cells are prone to accumulating lipid droplets, your bright intracellular structures. It is dependent on culture conditions and macrophage differentiatio. Oil red O staining will confirm. https://www.sciencedirect.com/science/article/abs/pii/S1388198121001098

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Gisela Beutner Many thanks for your kind attention and thoughtful reply to my observations.

1. Yes, the culture has been maintained.

2. Yes, the cells are growing well and responding to differentiation stimuli.

3. The medium appears to be normal, with full changes performed weekly depending on cell growth.

4. Yes — I plan to prepare a fresh batch of medium, one with a higher concentration of antibiotics and another without antibiotics, to monitor these structures more carefully under controlled conditions.

5. I am also testing the medium alone.

Malcolm Nobre Thank you for your attention and for your thoughtful response to my observations. The size and appearance of the structures suggest that it might indeed not be a contamination. However, their movement (at least to me) does not seem to be Brownian, but rather directional. I have previously observed small, dark, motile structures — which I interpreted as contamination — using this same observation method with Trypan Blue. Some of my colleagues also interpreted these structures as indicative of contamination. In any case, I will keep these cultures and perform experiments to determine whether they are contaminated or not, and I will surely keep your advice in mind.