Nis element imaging software

Calcein-AM (LifeTechnologies, Catalog Number C3099) was used to image the cells inside the microtumors during dose titration and drug screening as a validation approach. The Calcein-AM assay was carried out according to manufacturer’s protocol for Day 7 time points when possible. Images were then acquired with Nikon NIS-Element imaging software via Nikon Eclipse TS100 inverted fluorescent microscope, a Nikon B-2A FITC filter block (λ_excitation 465–495 nm bandpass, dichroic mirror 500 nm, λ_emission 500 nm long pass), EXFO X-Cite series 120 fluorescent source, and Q Imaging QICAM 12-bit Color Fast 1394 camera.

MKN45 cells were seeded onto coverslips placed in 12-well culture plates at 1 × 10⁵ cells/well and incubated for 24 hours. Subsequently, 1 μmol/L O-chlorin was added to the culture media and the cells were incubated for a further 4 hours before staining with organelle-specific fluorescent probes. Lysosomes were stained with 0.1 μmol/L LysoTracker Green (Invitrogen) at 37°C for 30 minutes, mitochondria with 0. 1 μmol/L MitoTracker Green FM (Invitrogen) at 37°C for 10 minutes, Golgi with 5 μmol/L NBD C6-ceramide at 4 °C for 30 minutes, and endoplasmic reticulum with 0.1 μmol/L ER-Tracker Green (Invitrogen) at 37°C for 30 minutes. After incubation, culture media were replaced with fresh medium to remove free dyes, and then the stained cells for observed, live for mitochondria and following fixation with 4% paraformaldehyde for lysosomes, Golgi, and endoplasmic reticulum. To visualize the localization, confocal laser microscopy (Nikon A1 confocal system Nikon Instech Co., Ltd.) was used and the obtained data were analyzed with NIS element imaging software (Nikon). Band-pass emission filters of 505-530 nm and 650 nm were used.

CTCs were fixed in 4% paraformaldehyde, permeabilized with phosphate buffered saline (PBS) plus 0.2% Triton X-100 and blocked for 60 min with 1% bovine serum albumin in PBS. Mouse anti-human pan-cytokeratin (C11) Ab (1:50, sc-8018, Santa Cruz Biotechnology, Heidelberg, Germany), anti-human CD45 Ab (1:50, #13917, Cell Signaling Technology, Danvers, MA, USA), anti-human TWIST1 Ab (1:50, sc-81417, Santa Cruz Biotechnology), anti-human EGFR (1:50, sc-373746, Santa Cruz Biotechnology), anti-human TRPM4 (1:50, HPA041169, Sigma Aldrich, St. Louis, MO, USA), and anti-human ZEB1 (1:50, sc-515797, Santa Cruz Biotechnology), followed by corresponding IgG Abs (Alexa Fluor^® 594, 1:100, Abcam, Cambridge, UK) were used to stain the CTCs. PureBluTM DAPI (#1351303, BioRad) labeled the nuclei. C2 Plus confocal laser scanning microscope (Nikon Instruments, Firenze, Italy) and NIS Element Imaging Software (Nikon Instruments) were used for the acquisition and processing of data. Epithelial CTCs required having a DAPI-positive nucleus with a diameter of 4 μm, cytokeratin staining surrounding 50% of the nucleus, and no CD45 expression.

Videos of free particles
in microtiter wells were made on a Nikon Ti confocal microscope (Nikon
Instruments Europe BV, The Netherlands) at a magnification of 20×
using an iXon Ultra 897 EMCCD camera (Andor, Belfast, UK) in bright-field
illumination conditions, using Nikon NIS-Element imaging software.
The positions of the particles were recorded in a field of view of
410 × 410 μm² at a frame rate of 33 Hz with
an exposure time of 5 ms. The particles were localized using phasor-based
localization, after which the x–y trajectories were used to calculate the mean squared displacement
over time using a sliding window algorithm. To discriminate between
unbound and bound states of particles, a threshold on the calculated
diffusion coefficient was set at 0.12 μm²/s.
Tracking of tethered particles in flow chambers was done on a custom-made
optical setup at a total magnification of 10× using a Grasshopper
camera (Point Grey Research Grasshopper3 GS3-U3-23S6M, 1920 ×
1200, pixel format: 8 raw, gain 10) in bright field illumination conditions.
The positions of the particles were recorded in a field of view of
659 × 493 μm² at a frame rate of 30 Hz with
an exposure time of 5 ms. The particles were localized using phasor-based
localization, after which the x–y trajectories were used to detect particle switching events using
a change-point detection algorithm, which has been described by Bergkamp
et al.^{14 (link)}