Hi all,
I am trying to optimization of MitoTracker™ Red CMXRos and Annexin V in alginate encapsulated cells via flow cytometer. What I am doing is that the beads are stained with Mito Tacker Red and then EDTA is used for the cell release. Once the cells released, Annexin V staining is continued and then flow cytometer run is done.
My question is that retain of Mito Tracker red staining after the 55mM EDTA treatment (5 mins @37 °C to release the cells from alginate and then continue staining of Annexin V. wondering if I use EDTA to release cells, can I lose Mito Tracker signal? or has anyone had any suggestions?
Looking forward to hearing all suggestions.
Cheers,
Hi Kadriye Guven
I quoted the following from Article MitoCeption as a new tool to assess the effects of mesenchym...
"Importantly to prevent such MitoTracker leakage, we found it necessary to avoid incubation of the MitoTracker-labeled MSCs with EDTA containing solutions.", it seems the author believes that EDTA treatment would cause leakage of MitoTracker.Another important issue is that Annexin V staining solution should have sufficient amount of calcium for Annexin V binding to the exposed PS on the cells' surface, so if you still have EDTA in the staining solution it might chelate the calcium required for binding Annexin V.
Mhd Yousuf Yassouf Thanks for the help. One more question, do you think it might be good to use EGTA or 55 mM sodium citrate rather than EDTA? or just after MitoTracker Red, I can take images on a Microscope instead of a Flow cytometer? I have another concern which is the effect of the dissolving solution on my cells. It might be hard to interpret my data.
Kadriye Guven I think the best way is to perform a preliminary experiment to test how different conditions affect the staining results.
Choosing the methods to perform the experiment totally depends on the design and purpose of the experiment.
Mhd Yousuf Yassouf actually, did a preliminary experiment to understand different conditions. However, I couldn't interpret it due to technical troubles. Thanks for all suggestions.
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