Hi!
I want to make a direct co culture experiment with mesenchymal stem cells from dental pulp and bone marrow. To distinguish between them, I want to use some fluorescent tracer (bought previously in my lab; diI, diO, diD). I have several questions:
- Solid formats (D-282, D-275 and D-307) could be used in living cells in culture? (In all papers I've found, they use Vibrant solutions forms). What could be the best incubation concentration?
- Fixation and/or permeabilization could alter fluorescent staining? I pretend to visualize cells by confocal microscopy and I would like counterstain with DAPI.
I know there are cell trackers but I already have these fluorescent tracers and I want to use them first if it is possible.
Many thanks in advance.
C.
5 uL per mL of solution (a maximum of 10^6 cells/mL) is the recommended concentration for Vybrant DiD. I personally found that DiD works better for most cell lines than DiO or DiI.
For your second question. I've fixed/permeabilized cells and then stained after with DAPI and it works great. I personally haven't fixed/permeabilized cells after staining with DiD, but I believe the staining should still work fine.
1) Lipophilic fluorescent dyes work with several protocols but their use is limited to short term experiments, If you want to examine cellular interactions between bone marrow MSC and dental pulp MSC during several cell divisions the concentrations of transient fluorescence dyes are reduced in each daughter cell. In this case I'd like to recommend stable transduction e.g. using adenoviral or lentiviral vectors containing fluorescence genes like GFP or mcherry/tomato. This way you'll have stable fluorescence during all subsequent passages of the MSCs.
2) Fixation/permeabilization of fluorophor-labeled cells for subsequent DAPI staining works but should be performed within a short period of time to avoid significant loss of cellular fluorescence.
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