I'm working with mesenchymal stem cells, and made an observation that I'm scratching my head to explain.
After spinning down cells and washing in PBS the cell pellet for WT cells is white, but the knockdown line I'm working with retains the pink color of the media.
Any ideas what could be causing this?
Hi! I have rather some guesses than the answer and would be interested to see other comments as well. Phenol red dye containing in the medium tends to stain any cells. Working on fluorescence imaging of living cells I observed Phenol Red fluorescence of different intensities depending on the cell line however the colour was not visible by eye. However, your particular modified cells might become more permeable to the dye or surface charge or other cell surface changes might lead to the more efficient dye-cell interactions. It may help to use Phenol red free media to avoid this as I do to exclude artefact fluorescence. Good luck.
Hi. Which kind of gene silencing did you use to obtain gene knockdown? These may help for finding a possible explanation.
Thank you for the responses. I'm using these cells for flow, so I'll check if the stain could be interfering with the fluorescence measurements. In addition, knocking down this gene is predicted to cause an increase in ROS, so maybe that could make the membranes more permeable?
The method I'm using for gene silencing is infection with a lentivirus carrying a vector coding for shRNA. The controls were infected with a non-targeting shRNA.
Maybe the membranes become more permeable after gene silencing. A significant increase of ROS pathway may influence mitochondrial homeostasis. Imbalance between the production of ROS and their oxidation has been reported to alter the membrane permeability and calcium homeostasis.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining