35 mm glass bottom dishes

For live cell imaging, cells were seeded onto 35 mm glass-bottom dishes (Greiner-bio-one, #627870) and the culture media were replaced to DMEM containing 10% FBS without phenol red (Sigma). Leica TCS SP8 inverted confocal microscope equipped with a Leica HCX PL APO × 63/1.4 oil CS2 objectives was used and cells were maintained in a chamber in 5% CO₂ at 37 °C during the experiments. The pinhole was adjusted at 2.0 airy units. The images were collected at 0.5 μm z-steps and 8–9 sections every 10 s.

Mel202 cells were transiently transfected with Halo-PERP, mCherry-SERCA2b and control vectors. Cells were seeded at a density of 4 × 10⁵ cells/well in 6-well plates and the following day were transfected with 1 μg of plasmid DNA and 2 μl of TurboFect in vitro transfection reagent (Thermo Scientific, Paisley, UK). For live-cell imaging, 3 × 10⁵ HeLa BAC Venus-PERP cells were seeded in 35-mm glass-bottom dishes (Greiner Bio-One, Stonehouse, UK) and transfected with 1 μg of mCherry-SERCA2b using 2 μl of TurboFect. For HaloTag pull-down, 2.5 × 10⁶ cells were seeded in 100-mm dishes (four dishes per transfection) and transfected using 5 μg of plasmid DNA and 10 μl of TurboFect. Cells were treated with 1 μg/ml BFA, 20 μM MG132, 100 μM CQ, or 1, 10 and 100 nM Baf A1 (all from Sigma-Aldrich).

Lysosomes was demonstrated using the LysoTracker™ Green DND-26 (Thermo Fisher Scientific). Cells were seeded in 35 mm glass-bottom dishes (Greiner Bio-One) at a density of 1 × 10⁵ cells/ml overnight and treated with CAP for 0–40 s at 70–80% confluency, the fresh media containing 0–100 μg/ml AuNPs was then replaced and incubated for 24 h or 48 h at 37 °C as indicated. After treatment, the cells were rinsed thrice with phosphate buffered saline and incubated with prewarmed (37 °C) LysoTracker-containing (50 nM) media for 5 min at 37 °C. Cells were then washed once with phosphate buffered saline and loaded with fresh phosphate buffered saline and observed using Zeiss LSM 510 confocal laser scanning microscope fitted with the corresponding filter settings as follows. LysoTracker™ Green DND-26, excitation wavelength: 488 nm, emission filter: 505–530 nm; AuNPs, excitation wavelength: 633 nm, reflection filter: 649–799 nm. Plan-Apochromat 63 × /1.4 Oil Ph3 was used as objective for all samples. To determine the level of AuNPs reflection, around 50 cells were randomly imaged for each treatment condition and the levels of reflection was analysed using the ImageJ and compared with other groups.

S2R+ cell culture, transfection, and generation of stable cell lines was done essentially as previously described [83 (link)]. For time lapse imaging, cells were plated in 35mm glass-bottom dishes (Greiner Bio-One GmbH). Expression of Separase activity sensors was induced by addition of 250 μM CuSO4 to the culture medium and cells were imaged 48 hours later.

Plasmids encoding Perceval, PercevalHR and pHRed were obtained from Addgene (Cambridge, MA). HCASMCs expressing PercevalHR or pHRed were transiently transfected using either FuGENE6 (Promega UK, Southampton, UK) or Promofectin (Promocell, Heidelberg, Germany) following manufacturer’s instruction. HCASMCs were seeded 24 h prior to transfection at a density of 1.25 × 10⁵ per well in 35 mm glass-bottom dishes (Greiner Bio-One, Gloucestershire, UK). The cells expressing Perceval and PercevalHR were used after 24–48 h of transfection, and cells expressing pHRed were also used after 24-48 h of transfection. The cells expressing PercevalHR based on a conventional 3rd generation lentivirus packaging system which has been described previously were used in PercevalHR in vitro calibration experiments (Yang et al., 2017 (link)).

An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (K101-100; BioVision, Milpitas, CA, USA) and CM-H2DCFHDA (C6827; Invitrogen, Waltham, MA, USA) were used for a cell death assay and cellular ROS imaging, respectively. In brief, SH-SY5Y cells (5.0 × 10⁵ cells/well) were seeded on 35-mm glass-bottom dishes (Greiner Bio-One, Kremsmünster, Austria). After being cultured in medium containing 5 μM Aβ₄₂ for 24 h, the cells were washed twice with PBS, and then incubated with annexin V-FITC and propidium iodide (PI) for 5 min at room temperature in the dark. For the detection of ROS, the cells (5.0 × 10⁵ cells/well) were seeded in 35 mm glass-bottom dishes and pretreated with CM-H2DCFHDA (10 μM) for 30 min at 37 °C in the dark, and then treated with 5 μM Aβ₄₂ for 3 h. The cells were washed twice with PBS. Fluorescent images were visualized immediately using a confocal laser scanning microscope (BZ-X700; Keyence, Osaka, Japan).

MCF10A cells were seeded in 35 mm glass bottom dishes (Greiner) and were subjected for live imaging 24 h after TGFβ treatment and transfection with ANGPTL4‐mCherry and Actin‐Chromobody SNAP plasmids using Lipofectamine 3000 according to manufacturer's instructions. SNAP‐Cell 647 SiR (1:2000, NEB, S9192S) was added, followed by 30 min incubation before imaging. Hoechst 33 342 was directly added (1:1000, Invitrogen, R37605) into the dishes for staining of the nuclei. Structured illumination microscopy (SIM) imaging was performed with an ELYRA 7 microscope (Zeiss) equipped with a 63  ×  1.4 Oil DIC objective and a Pecon incubation chamber, providing a stable environment for the samples at 37 °C and 5% CO2. All acquired imaging data were SIM processed using Zen 3.0 black edition and analyzed with Imaris 9.8.0.