- Place the virus-containing mixture to U2OS cells for infection.
- After 4 h of infection, remove the medium and replace it with
fresh D-MEM containing 10% FBS. - Culture the infected U2OS cells in DMEM containing 10%
FBS.
3.2.2 Preparation of the
Observed Cell Sample
Expressing iRFP-TRF1,
hnRNPA1-SNAPf, and the
TERRA Probe
- Culture the infected U2OS cells in DMEM containing 10%
FBS on glass-bottomed dishes. - Introduce plasmids of hnRNPA1-SNAPf/pcDNA3.1(+) and
NLS-GN-mPUMt-GC/pcDNA3.1(+) into the cells using
Lipofectamine LTX™according to the manufacturer’s proto-
col (seeNote3). - Incubate the cells for 4–6 h.
- Incubate the cells with 0.1μM SNAP-TMR for 30 min in a
CO 2 incubator to stain hnRNPA1-SNAPf. - Wash the cells with DMEM containing 10% FBS to eliminate
unbound SNAP-TMR. - Replace the cell cultivation medium with imaging medium.
- Place the sample onto the microscope stage after setting the
microscope for observations.
3.3 Microscopy
3.3.1 Construction and
Tuning of the Microscope
Setup
- Place optical components as Fig.3 shows *.
- Modulate the diaphragms’ height to make their centers identi-
cal to that of optical port of the microscope (H¼193.5 mm for
an Olympus IX 81; Olympus Optical Inc.).
Fig. 2Schematic of the principle of the present TERRA probe: (A) illustration of the probe, which consists of an
N-terminal EGFP fragment (GN); a PUM-HD mutant mPUMt that is modified to recognize an RNA of a
UUAGGGUU sequence, and a C-terminal EGFP fragment; (B) mechanism of the probe function to label
TERRA selectively (Reproduced from [12] with permission from Nature Publishing Group)
Spatiotemporal Imaging of Single Telomeric-Repeat Containing RNA 343