The number of vesicles transferred was quantitated using a vesicle transfer assay.20 (link),41 (link) Briefly, one flask of confluent TM cells was trypsinized, and half was labeled with Vybrant DiO dye (488 nm), while the other half was labeled with DiD dye (647 nm; ThermoFisher). After washing, fluorescently labeled cells were mixed 1:1, plated at 1 × 105 cells/mL, and incubated overnight. For NTM/GTM coculture assays, NTM (n = 5) cells were labeled with DiO and incubated with DiD-labeled GTM (n = 6) cells. Cells were then fixed and immunostained with CD44 monoclonal antibody and imaged by confocal microscopy. The total number of DiO (green) and DiD (red) cells were counted in each image. In addition, the number of TM cells containing at least five vesicles of the opposite color “transferred vesicles” was counted. For GTM/NTM cocultures, the percentage of green NTM cells containing red GTM-derived vesicles was calculated, and vice versa.
Protocol detail
Quantitative Vesicle Transfer Assay
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Assays
Cd44
Cocultures
Confocal microscopy
Monoclonal antibody
Red cells
Corresponding Organization : Oregon Health & Science University
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Variable analysis
independent variables
- Type of cells co-cultured (NTM vs. GTM)
- Number of DiO (green) and DiD (red) cells counted in each image
- Number of TM cells containing at least five vesicles of the opposite color ("transferred vesicles")
- Percentage of green NTM cells containing red GTM-derived vesicles, and vice versa
- Confluent TM cells used
- Cells labeled with Vybrant DiO dye (488 nm) and DiD dye (647 nm)
- Cells mixed 1:1 and plated at 1 × 10^5 cells/mL
- Cells incubated overnight
- Cells fixed and immunostained with CD44 monoclonal antibody
- Not explicitly mentioned
- Not explicitly mentioned
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