Nunc lab tek chambered coverglass

For immunodetection of ASMA, 1 × 10⁴ cells per cm² were seeded on Nunc Lab-Tek Chambered Coverglass (Thermo Scientific, Waltham, MA, USA) and cultured until 50% confluency. Cells were rinsed with PBS, fixed in 4% paraformaldehyde for 20 min at room temperature (RT), rinsed in PBS, and stored at 4 °C. Cells were permeabilized for 30 min at RT with 0.05% Triton X-100 in PBS complemented with 1% Bovine Serum Albumine (BSA) and 1% Normal Goat Serum (NGS) to reduce non-specific staining. Incubation with an antibody directed against ASMA (MAB1420; R&D System) at a dilution of 1/100 in PBS with 1% BSA and 1% NGS was performed overnight at 4 °C. Cells were washed three times in PBS, followed by incubation with Alexafluor 488 conjugated goat-anti-mouse IgG (Life Technologies, Saint-Aubin, France) at a dilution of 1/500 for 1 h at RT in darkness. After several washes, nuclei were stained with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; Life Technologies) at a concentration of 1/40,000 for 10 min at RT in darkness. Images were captured using a Nikon A1RSi confocal laser-scanning microscope (Nis Elements Confocal, Nikon, Amstelveen, The Netherlands).

Lung cancer cells (NCI-H1703) were maintained at 37 °C and 5% CO₂ in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum and 1% sodium pyruvate (Thermo Fisher Scientific). The lsFCS and lsSCS measurements were performed 24 h after seeding the cells on 8-well Nunc Lab-Tek chambered cover glass (Thermo Fisher Scientific). For membrane staining, stock solutions of CellMask Deep Red and CellMask Green plasma membrane stains (Thermo Fisher Scientific) were diluted with pre-warmed RPMI medium at a ratio of 1 : 3 × 10⁶ and 1 : 2.5 × 10⁴, respectively. The cell medium was exchanged with the dye solution and, 10 min later, the staining medium was again replaced by fresh and pre-warmed RPMI medium. Immediately afterwards, the experiments were started.

The labeled protein was diluted to 0.5 to 1 μM in buffer containing 10 mM of the nucleotide (GDP and GppNp from Jena Bioscience). The mixture was incubated for 2 hours at 37°C and further diluted at room temperature to 50 to 100 pM protein in buffer containing 1 mM nucleotide and bovine serum albumin (BSA) (0.1 mg/ml) (Sigma-Aldrich), and 30 μl was put on a coverslip (Nunc Lab-Tek Chambered Coverglass, Thermo Fisher Scientific BVBA) that was first incubated with BSA (1 mg/ml) and then washed twice with the sample solution. The background (needed for calculating E and S parameters, and for lifetime and PDA analysis) or scatter profile (needed for lifetime analysis) reference consisted of the same sample but without the protein or nucleotide. The small extra contribution of 1 mM nucleotide had a negligible effect. Measurements were performed at 22° or 37°C by placing the sample in a custom sample holder that was connected to a thermostatic water bath. When measurements were performed directly after mixing the protein and nucleotides, the closed/open ratio for the GDP state was higher. We attribute this to the presence of trace amounts of GTP in the GDP preparation. Therefore, we tested different premeasurement incubation conditions: overnight on ice and 2 hours at 37°C. We also tested whether measuring at 37°C instead of room temperature had an effect on the protein conformation.

The fluorescence and DIC imaging were performed using a Zeiss LSM 710 laser scanning confocal microscope, equipped with a 63× oil immersion objective (Plan-Apochromat 63×/1.4 oil DIC M27) and a Zeiss Primovert inverted microscope. Samples were prepared and imaged using tween-coated (20% v/v) Nunc Lab-Tek Chambered Coverglass (ThermoFisher Scientific Inc.) at room temperature (22 ± 1 °C) unless otherwise noted, with ~ 1% labeled protein samples within the mixture of unlabeled proteins. All the samples were allowed to equilibrate in the chambered coverglass for ~30–45 min before imaging. For Alexa488-labeled samples, the excitation and emission wavelengths were 488 nm/503–549 nm; for Alexa594-labeled samples, the excitation and emission wavelengths were 595 nm/602–632 nm. Fluorescence recovery after photobleaching (FRAP) experiments were performed using the same confocal set up. The images and data were analyzed using Fiji software [70 (link)] and the FRAP curves were plotted and analyzed using origin software (OriginPro 2018).