To begin, take the co-cultured MSC-Mito-GFP and ARPE19-Mito-RFP cells and remove the medium from the 24-well plates. Wash all cells once with 500 microliters per well of 4%poly formaldehyde. Add 500 microliters of fresh 4%poly formaldehyde and fix the samples for 20 minutes.
Then remove the fixative and wash the samples three times for 10 minutes each with DPBS. After removing the PBS, add 500 microliters per well of blocking solution and incubate for one hour at room temperature. Then remove the blocking solution.
To prepare the phalloidin staining solution, dilute the 400x storage solution to 1x with PBS. Add 200 microliters of phalloidin staining solution to each well and incubate for one hour at room temperature. Then recover the staining solution and wash the samples for 10 minutes with DPBS.
After removing the PBS, pick out the cover glass with a syringe needle and allow it to air dry. Apply a small amount of mounting medium onto the slide and carefully flip the cover glass on the slide to ensure that the medium evenly coats the entire surface. Seal the edges with nail polish.
Tunneling nanotubes or TNT structures were observed, establishing intercellular connections between both similar and different cell types. Mitochondrial transfer was observed in ARPE-19 cells containing green dotted fluorescence from MSCs and in MSCs containing red dotted fluorescence from ARPE-19 cells. Super resolution imaging confirmed the formation of TNTs and detailed mitochondrial transfer via TNTs.
Quantification showed MSCs were significantly more capable of donating mitochondria compared to a ARPE-19 cells.
