35 mm glass bottom dish

Expi293F cells (Thermo Fisher Scientific, Waltham, MA, USA) were maintained in Expi293 Expression Medium (Thermo Fisher Scientific) under 8% CO₂ at 37°C on an orbital shaker platform. For cDNA transfection, cells were plated on a 35-mm glass-bottom dish (AGC Techno Glass Co., Ltd., Yoshida, Japan) coated with poly-L-lysine (Sigma, St. Louis, MO, USA) the day before and transfected with the expression plasmids using Lipofectamine 3000 (Thermo Fisher Scientific). After overnight culture, cells expressing fluorescent protein-fused glycosyltransferases were observed using a super-resolution microscope.

Ax2 cells expressing the ratiometric fluorescent indicator (fusion protein of Flamindo2 and mRFP) were maintained in a 90 mm plastic dish at 22 °C in HL5 medium supplemented with 50 µg/mL hygromycin (Wako, Osaka, Japan) and 16 µg/mL G418 (Wako, Osaka, Japan). Cultured cells were suspended by pipetting on the dish and split into a new dish at an approximate ratio of 1:6 every 24 h, and the cells were maintained at an almost constant cell number for a long time. Development of the cells was initiated at t = 0 h by inducing starvation after washing gently thrice with the development buffer (5 mM Na₂HPO₄, 5 mM KH₂PO₄, 1 mM CaCl₂, 2 mM MgCl₂, pH 6.4) to avoid detaching from the dish surface. These cells were suspended in the developmental buffer and the cell number was counted using Countess-II (Thermo Fisher Scientific, Massachusetts, USA). Cells (1.8 × 10⁶) were plated in a 35 mm glass-bottom dish (AGC Techno Glass, Shizuoka, Japan) in 1.6 mL of developmental buffer.
In the fluorescence imaging, two LED light sources with center wavelengths of 470 nm and 525 nm (SOLIS-470C and SOLIS-525C, Thorlabs, Newton, NJ) were used for the excitation of Flamindo2 and mRFP, respectively. The sample dish was placed in a stage-top chamber at 22 °C and imaged by the AMATERAS1.0 system for 16 h from t = 4 h to t = 20 h. The imaging interval of the image sequence was 30 s, resulting in 1,921 frames for 16 h observation. At each time frame, the fluorescence of Flamindo2 and mRFP were sequentially imaged by switching the LEDs and emission filters. The excitation intensities of the two LEDs were 13.4 mW/cm² (470 nm) and 15.4 mW/cm² (525 nm) in the sample plane, respectively. Both fluorescence signals were detected with a 1.3 s exposure with 8 × gain.