Glass bottom microwell dishes

[Ca²⁺]_i was measured as previously described32 (link). Briefly, serum-starved Caco-2 cell monolayers on glass-bottom microwell dishes (MatTek; Ashland, MA) were incubated with Fura-2AM (10 μM) in 0.5% pluronic acid for 30 min. Fura-2-loaded cells were alternately excited at 340 or 380 nm using a PC-driven hyper-switch (Ionoptix; MA, USA). Ratios were collected every second at 510 nm using a Dage MTI iCCD camera and Ionwizard software (Ionoptix). [Ca²⁺]_i was calculated using the following equation: [Ca²⁺]_i = K_d (R−R_min) (S_f2)/(R_max−R) (S_b2), where R is the 340/380 nm ratio, R_min and R_max are the minimum and maximum ratios determined in Ca²⁺-free and saturated Ca²⁺ solutions, respectively, S_f2/S_b2 is the Ca²⁺ free/Ca²⁺-replete ratio of emissions at 380 nm excitation, and K_d is the dissociation constant for Fura-2. R_min, R_max, S_f2, and S_b2 were determined by increasing the Ca²⁺ permeability of Caco-2 cells with ionomycin (10 μM), and perfusing cells with a high-Ca²⁺ (10 mM) or Ca²⁺-free (10 mM EGTA) solution. The in situ apparent dissociation constant (K_d) for Fura-2 used in this study was 224 nM. Eight to ten cells in each monolayer were analyzed simultaneously, and the experiments were repeated in 3–5 monolayers.

Concentrated suspensions of three series of preparation: (1) fresh, (2) 2.5% glutaraldehdye-fixed (Electron Microscopy Sciences, Hatfield, PA, USA) and (3) fixed-Nile Red (Sigma-Aldrich Corp., St. Louis, MO, USA) (5 micrograms/mL) treated cells were deposited onto the coverslip areas in glass bottom microwell dishes (MatTek Corp., Ashland, MA, USA) and examined by confocal microscopy using a model TCS SP5 system coupled to a DMI 6000 inverted microscope equipped with a 100× objective lens (Leica Microsystems, Exton, PA, USA) in the x,y,z imaging mode and fluorescence scanning mode with excitation from the 488 nm line of an Argon laser. Images were collected in data sets of two channels (500–550 nm and 660–720 nm) for fresh- and glutaraldehyde-fixed cells or in three channels for Nile Red-treated cell suspensions (500–550 nm, 570–620 nm and 660–720 nm) and examined as maximum projections (8–12 micrometers deep) in separate and graphically overlaid image channels. Fluorescence emission scans were performed from 500–750 nm using a 15 nm detector window and frame averaging of selected focal planes. DIC images were taken using the same method for laser-scanning confocal microscopy; however, the transmitted light channel (non-confocal) was employed here.

The ALS CellCelector™ (Automated Lab Solutions, Germany) was utilized to automatically image, enumerate and select CTC based on immune phenotype. Briefly, after CD45 depletion, the enriched CTCs were stained live for the following cell surface antigens: EpCAM (rabbit anti EpCAM ab, clone D4K8R, Cell Signaling, 1:50 dilution) followed by anti-rabbit Alexa 568 and CD45 (mouse anti CD45-Pacific blue ab, clone H130, Biolegend, 1:50 dilution). Cells were subsequently plated on glass-bottom microwell dishes (Mat Tek Corporation) for automated scan and live imaging. CTCs were visualized under the microscope and EpCAM^pos/CD45^neg cells were isolated as single or as pools of up to 50 cells by the robotic arm of the instrument and processed for ddPCR. To identify EpCAM^neg/CD45^neg cells as bona-fide CTCs, visual inspection was performed based on established criteria including larger size, greater nuclear to cytoplasmic ratio, as well as distinct nuclear morphology^{43 (link)}. For single VCaP, 22Rv1 or CWR-R1-D567 analysis, cells were plated on glass-bottom microwell dishes, and individual cells were marked under bright field before microcapillary mediated mechanical suction (Fig. S2e). Effective single-cell picking was confirmed by visual inspection of the marked positions before and after picking (Fig. S2d). Single cells were processed for ddPCR.

10 × 10⁴ HeLa cells were seeded in complete medium on glass-bottom microwell dishes (MatTek Corp.) for 48 h at 37°C in 5% CO₂. Cells were washed with 1 × PBS, and DBI (1 μM), Temoporfin (10 μM), or an equivalent amount of DMSO (0.02% v/v), dissolved in complete medium and were added to cells for 1 h at 37°C in 5% CO₂. Then, the cells, when required, were photo-irradiated using an EVOS® FL cell imaging system equipped with an adjustable-intensity LED cube (Ex: 470/22 nm) operating at 30 mW cm⁻² at various time intervals.

Whole zebrafish embryos were mounted laterally in 0.5% low-melt agarose (Apex Bioresearch Products) inside glass-bottom microwell dishes (MatTek Corporation). Imaging was performed on a Nikon Eclipse Ti confocal microscope, with excitation by 488nm (eGFP) and 561nm (tdTomato and dsRed) lasers. All imaging was done at 28.5°C, with embryos staged as described previously [46 (link)].

Breast cancer cell lines were grown on sterilized glass-bottom microwell dishes (MatTek Corporation) for 2 days and then fixed and permeated. Staining was performed as described in Supplementary material.