Hi
I have stem cells encapsulated in sodium alginate and I want to stain them for Fluorescence microscopy. What is the best concentration of the DAPI and PI ?
Do you have any protocol for that?
Hi Maryam Rahimi
The use of DAPI (4',6-diamidino-2-phenylindole) and PI (Propidium Iodide) are common for fluorescent staining of cells. Here's a general protocol you can follow, but please keep in mind that you may need to adjust the concentrations based on your specific experimental conditions and needs:
Note: It's important to protect your samples from light as much as possible during the staining process and subsequent handling to prevent photobleaching of your fluorescent dyes.
As always, remember to include appropriate controls in your experiment, and check the compatibility of your encapsulation material (in this case, sodium alginate) with the staining protocol and the imaging techniques you plan to use. Good Luck!
Both DAPI and PI preferably stain dead cells. DAPI is impermeable to live cells unless it is used in higher concentration. For the nuclear staining of fixed cells 300nM DAPI is sufficient with 15-20 minutes incubation (for hydrogel encapsulated cells). In Calcein AM/PI LIVE/DEAD assay usually 1-2uM PI is generally used to stain cells with compromised membrane. Washing with 1x PBS is necessary for the removal of excess dye adsorbed on alginate surface. For imaging Z-stack fluorescence microscopy will give proper in depth images of your encapsulated cells.
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