In cell analyzer 6000

HEK293T cells were treated with 1× and 3× amounts of lentivirus expressing either DnaJB8–mClover3 or mClover alone were plated at 300,000 cells per well in media (10% FBS, 1% Pen/Strep, 1% GlutaMax in Dulbecco’s modified Eagle’s medium) in a 6-well glass bottom plate (Cellvis, P06-1.5-N). After 30 h, cells were stained with Hoescht33342 at a final concentration of 2 μg/mL in cell media for 30 min at 37 °C and 5% CO₂. The plate was placed on an IN Cell 6000 Analyzer (GE Healthcare) with a heated stage and 50 fields of view were imaged under 4′,6-diamidino-2-phenylindole (DAPI) and FITC channels at ×60 magnification (Nikon ×60/0.95, Plan Apo, Corr Collar 0.11–0.23, CFI/60 lambda). Images were exported as TIFF files for downstream analysis. DnaJB8–mClover3, mClover3, and WT HEK293 cells were plated and imaged in triplicates. Total cell counting was done using the CellProfiler v3.0 software^{50 (link)} by selecting for DAPI (total cells in acquired images) and mClover3 (total expressing cells in acquired images). Puncta-containing cells were counted manually by two different observers and the data were reported as the average with a standard deviation between both observers. Expression of Clover and DnaJB8–Clover in 1× and 3× cell lines was quantified from Western blot analysis and by fluorescence intensity of Clover quantified using ImageJ^{51 (link)}.

Cell cultures were washed twice with DPBS, then fixed with 4% (w/v) paraformaldehyde (Polysciences) for 10 min and permeabilized for 10 min with 0.1% (v/v) Triton X-100 (Thermo Fisher). The cells were then blocked with 3% (w/v) bovine serum albumin (Sigma) for 30 min, followed by incubation with the primary antibodies for 1 h. After three DPBS washes, the cells were incubated with secondary Alexa Fluor–conjugated antibodies (Invitrogen) for 1 h, followed by four DPBS washes. The nuclei were counterstained with DAPI (Invitrogen). The cells were then examined and images captured by confocal microscopy using the TCS SP8 (Leica) microscope using the Las X (Leica) imaging software or analyzed using a high-throughput screening IN Cell 6000 analyzer (GE Healthcare). All images from independent, but identical, experiments were acquired under the same conditions, and laser intensity levels were maintained constant throughout all experiments to reduce technical variability.

2.5 × 10⁵ primary cells were seeded in 24-well plate which was placed a sterilized glass slide in every well, incubated for 24 h, and fixed with 4% paraformaldehyde for 30 min. CK8/18, TTF1, NapsinA antibodies (Rat anti-human, 1:50; Santa Cruz Biotechnology) was incubated at 4 °C overnight, and then FITC-conjugated rabbit IgG antibody was used. Cells with green fluorescent signals in the nucleus and cytoplasm were counted as positive expression cells. The In Cell 6000 Analyzer (GE) was used for detecting the fluorescence.

A total of 8000 primary cells were suspended in 0.33% basal medium eagle agar supplemented with 10% FBS. The cells were maintained at 37 °C, 5% CO₂ for 2–3 weeks, and cell colony numbers were counted by In Cell 6000 Analyzer (GE).

2D NP cellular internalization was assessed using fluorescent dye (propidium iodide) loaded NP treatments, visualized with Zeiss LSM 710 confocal microscope to determine cellular localization and In Cell 6000 Analyzer (GE healthcare, Amersham, UK) for high throughput analysis of NP containing PI frequency. See supplementary materials for additional information.
3D NP internalization was assessed using flow cytometry conducted using an Amnis^® CellStream^® benchtop flow cytometry system equipped with 375 nm, 405 nm and 488 nm lasers, a dedicated side scatter laser (785 nm) and dedicated forward scatter LED (450 nm) coupled to a filter stack for spatial separations of photons (456/51, 528/46, 583/24, 611/31, 702/87 and 773/56 nm). 3D cell cultures were treated with NP treatments loaded with fluorescent dye propidium iodide for 48 h, 24 spheroids were used per treatment. Spheroids were collected and disaggregated via accutase (Sigma Aldrich; A6964-500mL) incubation for 10 min at 37 °C with mechanical pipetting every minute. Cells were pelleted and resuspended in flow cytometry buffer (1× dPBS supplemented with 3% FBS, 2mM EDTA and 2mM NaN₃) and nuclei were co-stained using Hoescht stain (Thermo Fisher Scientific, H3570). Samples were run at 14.64 μL and fluorescence was measured on channels A2 (405/528 nm) for Hoescht and C6 (488/702 nm) for PI.