H tet cy5

Trans-Cyclooct-2-en-L-Lysine (TCO*A; #SC-8008) was purchased from SiChem (Bremen, Germany). Pyrimidyl-Tetrazine-Alexa Fluor 647 (Pyr-Tet-AF647; #CLK-102), Pyr-Tet-ATTO-643 (Pyr-Tet-ATTO643; #CLK-101), H-Tet-Cy3 (#CLK-014-05), and H-Tet-Cy5 (#CLK-015-05) were purchased from Jena Bioscience (Jena, Germany). SNAP-Surface^® Alexa Fluor^® 488 (BG-AF488; #S9129S) was purchased from New England Biolabs. 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX; #1044) and Kainate (KA; #0222) were purchased from Tocris. l-Glutamic acid monosodium salt (Glu; #G1626) and doxycycline (#D1822) were purchased from Sigma.

H-tet-Cy5 was purchased from Jena Bioscience, Jena, Germany # CLK-015-05. Absorbance and emission spectra of Pyr-tet-ATTO643 were recorded in quartz glass cuvettes using an FP-6500 spectrofluorometer (Jasco). Excitation wavelength was positioned over absorption maxima, and spectra were recorded at constant 25°C stabilized via Peltier thermocouple. Time-dependent fluorescence intensities were measured in quartz glass cuvettes using an FP-6500 spectrofluorometer (Jasco). An increase in relative fluorescence for determining the turn-on ratio was measured after performing a click-reaction in cuvette applying 25 μM TCO^∗ and 1 μM dye solutions.

The cells in the T25 flask were detached in 5 ml new media 7 h after transfection. 300 μl of the cell solution was added to 300 μl new media in each well of the chambered cover glasses. The cells were incubated for another 12 h at 37°C and 5% CO₂. At the start of the experiment 10 μl of TCO*A (SiChem #SC‐8008) solved in 1 M HEPES was added with the final concentration of 250 μM to start the translation of CD55‐TAG‐mTurq^NTF. Confocal images were taken with a Zeiss LSM 980 Airyscan 2 and a HC PL APO CS2 63×/1.40 OIL objective. An 405 nm excitation laser with a 464–499 nm detection range was used for the mTurq‐channel, an 514 nm excitation laser with a 534–587 nm detection range for the mCitr‐channel and an 639 nm excitation laser with a 657–693 nm detection range for the Cy5‐channel. To cover a larger region of interest a tile scan was performed. The images were acquired with a 219 nm pixel size. An automated time series was performed in order to get images at periodic time points over 24 h. For live‐cell imaging via click labeling H‐Tet‐Cy5 (Jena Bioscience #CLK‐015‐05) was used. The wells were washed once with CGM and then incubated with 300 μl CGM and 1.5 μM Tet‐Dye for 10 min at 37°C. After a final washing step with CGM the wells were ready for imaging.