For single live-cell microscopy, SW480 and SW620 cells were transfected with a REV-ERBα-VNP plasmid using X-tremeGENE HP DNA Transfection reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. REV-ERBα-VNP positive cells were sorted via fluorescence-activated cell sorting and cultured under normal growth conditions until use. For the experiment, cells were plated in μ-Slide 8 Well Glass Bottom dishes (ibidi, Martinsried, DE). Time-course live-cell microscopy was performed using a CSU-X spinning disc confocal microscope (Nikon Corp., Tokio, JP). Pictures were taken and fluorescence intensity was determined every 30 min for 38 h.
μ slide 8 well glass bottom dishes
Manufactured by Ibidi
The μ-Slide 8 Well Glass Bottom dishes are a product offered by Ibidi. The dishes feature 8 individual wells with a glass bottom. The core function of this product is to provide a substrate for cell culture and microscopy applications.
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Lab products found in correlation
X tremegene hp dna transfection reagent, by Merck Group (1 mentions)
Hoechst 3342, by Thermo Fisher Scientific (1 mentions)
Albumin fraction 5, by Carl Roth (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Nabh4, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
2 protocols using μ slide 8 well glass bottom dishes
1
Live-Cell Imaging of REV-ERBα Dynamics
2
Immunofluorescence Assay for Confocal and STED Microscopy
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Immunofluorescence assays (IFA) of parasites for confocal and STED microscopy were performed as described previously in detail [83 (link)]. In brief, parasites were seeded to concanavalin A (Sigma-Aldrich) coated μSlide 8-Well glass bottom dishes (Ibidi), fixed the next day with 4% PFA (Electron Microscopy Sciences) in PBS (Gibco, Thermo Fisher Scientific) for 20 min at 37°C, treated with 0.1% Triton X-100 (Sigma-Aldrich) and 0.1 mg/ml NaBH4 (Sigma-Aldrich) PBS solutions for permeabilization and quenching of free aldehyde groups. Blocking as well as incubation of primary and secondary antibodies (S3 Table ) was performed with 3% w/v Albumin Fraction V (Roth) in PBS. Hoechst 3342 (Thermo Fisher Scientific) was added during secondary antibody incubation at a dilution of 1:1000. Unbound antibodies were washed off with 0.5% Tween-20 (Roth) in PBS.
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