Cell observer sd

Confocal microscopy for live-cell imaging was performed on a setup based on the Zeiss Cell Observer SD utilizing a Yokogawa spinning disk unit CSU-X1. The system was equipped with a 1.40 NA 63x Plan apochromat oil immersion objective from Zeiss. The setup was heated to 37 °C and a CO₂ source was provided to keep the atmosphere at 5% CO₂ during the measurements. The resulting images were processed with the Zen software by Zeiss. Cell spheroids were imaged in brightfield mode with a tungsten-halogen lamp with 682.0 μW and 200 ms exposure time. For fluorescence images, Hoechst 33342 was excited with a 405 nm laser at 11.2 μW intensity for 200 ms and Alexa Fluor 546 of the secondary YAP antibody with a 561 nm laser at 70.7 μW intensity for 1000 ms. Images with ATTO-633 dye were taken with a 639 nm laser at 5.5 μW intensity and 200 ms exposure time. Each image consisted of 120 to 160 pictures, depending on the spread of the spheroid over time, and has an 11 frame z-stack with a 1.5 μm distance between z-planes. In the excitation path a quad-edge dichroic beamsplitter (FF410 /504/582/669-Di01-25x36, Semrock) was used. Band-pass filters 525/50 and 690/60 (both Semrock) were used in the detection path. Separate images for each fluorescence channel were acquired using two separate electron multiplier charge coupled devices (EMCCD) cameras (PhotometricsEvolve^™).

NIH-3T3 cells (cell line collection, IIET PAS) were grown in DMEM (4.5 % of glucose) containing 10 % FBS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), l-Glutamine (2 mM), β-mercaptoethanol (9 µM). For immunofluorescence cells were grown to 90–100 % confluency on sterile coverslips placed in 24-well plate. Cells were cultured in serum-free medium for 24 h before fixation to promote cilia formation (Pitaval et al. 2013 (link)). Cells were fixed in 3.7 % formalin in PBS and incubated for 10 min at room temperature, permeabilized with 0.1 % Triton X-100 in PBS (10 min), washed with PBS and blocked for 1 h with PBS containing 1 % FBS. Cells were then incubated for 1 h at RT with primary (Ab285: 1/100; mouse monoclonal against acetylated α-tubulin: 2 μg/ml) and secondary (Cy2 conjugated donkey anti-rabbit: 15 µg/ml, Cy5 conjugated goat anti-mouse: 15 µg/ml) antibodies, respectively. Control reactions were carried out without primary antibodies. Labeled cells were mounted in moviol based mounting medium containing DAPI (2 μg/ml) as a nuclei marker. Cells were imaged by spinning disc confocal microscope Zeiss Cell Observer SD equipped with Yokogawa CSU-X1A 5000 unit with the use of 63 × oil-immersed Plan-Apochromat objective (NA 1.4). Z-stacks across entire cells were acquired at Z increments of 0.4 μm. Images were processed with the use of ImageJ (NIH).

HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS and 1% PenStrep. They were seeded into ibiTreat µ-slides (ibidi) at a concentration of 5000 cells per well in 300 µL of DMEM. The day after seeding, cells were incubated with particles for 24 h and for the lysosome experiments also with 8 µL of CellLight Lysosomes-GFP (Thermo fisher scientific, Waltham, MA, USA). The particles were labeled with Atto633-NHS overnight and then prepared as for the characterization described above. For internalization experiments cells were stained with WGA488 (Thermo fisher scientific, Waltham, MA, USA), and washed with DMEM prior to imaging. For imaging we used a spinning disk microscope (Zeiss Cell Observer SD with a Yokogawa spinning disk unit CSU-X1). Lysosome-GFP and WGA were excited with a 488 nm laser and the particles with a 639 nm laser. Band-pass filters 525/50 and 690/60 (both Semrock) were used in the detection path for Lysosome-GFP/WGA and the particles, respectively.