Dear fellow researchers:
I am looking for the best way to visualize adipocytes, which have been transferred from one mouse to another, and assess cell viability.
Volumes of 1-2 ml are obtained from one mouse and DiI stained before transfer and subcutaneous implantation in a second syngeneic mouse.
After a few weeks the adipose tissue is explanted.
Now I would like to assess whether the excised adipose tissue is made up of the previously transferred cells. This is what the initial DiI staining (red) is for. In addition I was thinking about cutting the tissue in 2-3 mm thin sections for staining with a) Hoechst for cell nuclei (blue) and Calcein AM to assess viability (green).
Does anyone have any experience with the combination of the above mentioned stains?
Can you recommend any specific products?
What would your suggestions for alternative methods and/or stains be?
Thank you!
Hi,
that sounds like an interesting approach.
Though Dil is freqently used as a quite stable tracer dye, make sure that it remains stable for the duration of the experiment and is not metabolized by the cells or diffues into the recipient mouse, e.g. by running appropriate controls. If stability proves to be a problem, consider a transgenic approach, e.g. transform adipocytes with a fluorescent protein marker (e.g. GFP or tdTomato). This can be done either stably (elaborate, but convenient as soon as established) or with an episomal vector (preferrable if you plan to do only a few experiments) for long-term expression and labelling, respectively.
Best regards
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