Imaging of glycoforms by transmembrane FRET

 Transfect cells of your choice with the plasmids using the FuGENE HD transfection reagent, according to the manufacturer’s protocol (see Comment).

 Seed the cells on cover glasses (12-mm diameter) placed in 24-well plates in appropriate medium and incubate in the absence or presence of 50 μM Ac₄ManNAz for 2 days to incorporate azide-labeled sialic acid into the cellular glycans (see Comment).

 Wash the cells with PBS(−).

 Fix the cells with 3% paraformaldehyde in PBS for 20 min at room temperature.

 Wash twice with PBS.

 Add 500 μL of 3% BSA in PBS per well and incubate for 30 min at room temperature.

 Prepare click reaction solution (250 μL per sample finally). Add 10 μL of Click-iT reaction buffer (component A) and TAMRA-alkyne stock solution to 230 μL of PBS and mix well (final concentration of TAMRA-alkyne: 1 μM).

 Add to the fixed cells.

 Add 2 μL of CuSO₄ solution (component B) and shake the plate gently.

 Add 4 μL of component C and shake the plate gently.

 Wait for 2–3 min, but not longer than 5 min, before proceeding to the next step.

  Add 4 μL of component D and shake the plate gently. The solution turns bright orange.

 Incubate for 30 min at room temperature (protect from light).

 Wash three times with PBS.

 Wash three times with PBS.

 Observe the fluorescent images by laser scanning confocal microscopy. To measure FRET, excite the sample at 488 nm and measure the fluorescence intensities of the donor (GFP) and acceptor (TAMRA) using an appropriate filter set (see Notes).

 Seed the cells expressing GFP-tagged protein onto a glass-bottom dish in appropriate medium and incubate in the absence or presence of 100 μM Ac₄ManNAz for 2 days.

 Wash the cells with KRB buffer or other appropriate buffers.

 Label cell surface azido sugars (SiaNAz) with 50 μM DIBO-555 in KRB buffer for 5 min at 37°C or for 1 h at 4°C. Place 100 μL of staining solution as to cover the cells on the glass position of the glass-bottom dish.

 Wash the cells with KRB buffer three times.

 Add pre-warmed KRB buffer and acquire fluorescent images using laser scanning confocal microscopy equipped with an incubator to maintain the cells at 37°C throughout the experiments. To measure FRET, excite the sample at 488 nm and measure the fluorescence intensities of the donor (GFP) and acceptor (Alexa Fluor 555) using an appropriate filter set (see Notes).