In addition, co-expression of these internalized receptors with a lysosome marker (LysoTracker Red DND 99; Figure 1B) indicated that DOR-eGFP receptors were targeted to the lysosomal compartment.
Several maternal mRNAs, such as vasa [1], nanos1 [3], dead-end (dnd) [4], and Tudor domain-containing protein 7 (tdrd7) [5], are known to be localized to the germ plasm (hereinafter called germ plasm mRNAs).
Then, the cells were incubated with 1 μM acidic indicator, LysoSensor Green DND 189 (Invitrogen) for 30 min in 37 °C.
To confirm these findings, we next used the acidotropic probe (p Ka = 5.2) LysoSensor Green DND 189.
LysoTracker Green DND 26 was added at a final concentration of 40 nM and incubated for 10 min.
A pH-sensitive fluorescent dye LysoSensor DND 198 (Molecular Probes, Inc., Eugene, OR, USA) was used to determine the intracellular lysosomal pH in living cells.
For lysosomal acidification assay, the cells were incubated with the 1.0 μM DND Green-189 (Invitrogen, L7525) for 30 min at 37 °C.
LysoTracker Red DND-99 (0.3 μM; L7528, ThermoFisher) was used to stain the lysosome compartments.
Monodispersed detonation nanodiamonds (DNDs) (5 10 nm) were used throughout (New Metals & Chemicals Corporation, Tokyo, Japan).
SiNPs treatment decreased the fluorescence intensity of Green-DND 189 (Fig. 6c, S2D), suggesting an increased lysosome pH.
The acid vesicular compartment was also detected by vital staining with LysoTracker Red DND-99 (Invitrogen) for 30 min.
