Nunc lab tek 2 8 well chamber slide

Daoy 30Q-RFP cells or Daoy 82Q-RFP cells were grown in culture and plated onto Nunc Lab Tek II 8-well chamber slides (ThermoFisher Scientific 154,534; Waltham, MA). The state of ATXN1 aggregation during the induction and inhibition of autophagy was determined using an autophagy assay kit (AbCam ab139484; Cambridge, MA). Briefly, 18 h following plating, cells were treated for 24 h with 500 nM rapamycin, dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich D2650;St. Louis, MO), and 60 µM chloroquine, dissolved in distilled deionized water, DMSO vehicle control or left untreated. Cells were stained for autophagy following the kit instructions and imaged on a FLoid Cell Imaging Station (ThermoFisher Scientific). Counts were performed from two randomly selected non-overlapping fields per chamber from each of three independent experiments.

Nunc Lab-Tek II 8-well Chamber Slides (Thermo Fisher Scientific, New York, NY, United States) were seeded with 30,000 Huh7 cells per well 24 h before infection. 10,000 WT or s20(-) salivary gland sporozoites suspended in 100 μl DMEM-complete were added and allowed to settle for 1 h at RT. P. berghei S20 was immunostained to investigate the expression of S20 during liver stage development. Here, infected hepatoma cells were incubated at 37°C for 1 and 3 h after infection prior to fixation with 4% paraformaldehyde and staining with monoclonal anti-HSP70 antibody (Tsuji et al., 1994 (link)), anti-S20 peptide antiserum, and Hoechst 33342 (Invitrogen, Carlsbad, CA, United States). Phenotypic analysis of parasite liver stage development was monitored by IFA in Huh7 cells 48 h post-infection at 37°C. The infected cells were permeabilized with 0.2% Triton-X 100 (Roth, Karlsruhe, Germany) and analyzed 48 h later after fixation with 4% PFA by staining with mouse anti-HSP70 antibody (Tsuji et al., 1994 (link)) followed by goat anti-mouse Alexa Fluor 488 antibody (Invitrogen, Carlsbad, CA, United States) and Hoechst 33342 (Invitrogen, Carlsbad, CA, United States). Slides were mounted with Fluoromount-G (SouthernBiotech) and imaged by fluorescence microscopy (Zeiss Axio Imager Z2). At least 20 intracellular parasites per sample were visualized.

We plated 50 k cells per well in complete RPMI lacking phenol red (ThermoFisher Scientific) in Nunc Lab-Tek II 8-well chamber slides (ThermoFisher Scientific #154534). We recorded time-lapse videos using NIS Elements II and an inverted live-cell microscope (Nikon, Melville, NY, USA) equipped with a cell incubation chamber that provided CO₂, heating, and humidity control. To visualize cell permeability, we added 0.5 μM Sytox Blue to the media. For each experiment, we tested five conditions concurrently: RodTox alone, RodTox+5 mM glycine, 5 mM glycine alone, media alone, and 1% Triton X-100. Following imaging, we sampled 50 μl of supernatant from each well to perform an LDH release assay as described above. For imaging of mitochondrial activity, we treated cells with 200 ng/ml of tetramethylrhodamine methyl ester perchlorate (TMRM, Life Technologies, Carlsbad, CA, USA) for 30 min before replacing with stimulation conditions. For nucleic acid dye staining kinetics experiments, we added 1 μM Sytox Blue, 1 μM propidium iodide (Sigma), and 1 μM ethidium bromide homodimer (Life Technologies) as indicated.

Cells were seeded in Nunc Lab-Tek II 8-well chamber slides (Thermo Fisher Scientific) or on glass coverslips in 24-well plates, coated with 0.1% gelatin. Cells were fixed by 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) and stained using primary and secondary antibodies and DAPI (Calbiochem, Merck Millipore) counterstaining, as reported [16 (link)]. Imaging was performed on the Zeiss Axio Observer.Z1 (Carl-Zeiss) equipped with the AxioCam MRm. Images were acquired using Zen 2.6 Pro (Blue) software, Carl Zeiss Canada Ltd, Toronto, ON, Canada.