The indicated cell lines were seeded in appropriate densities in sterile 96‐well plates (Greiner Bio‐One, Kremsmünster, Österreich) one day prior to stimulation with the indicated concentrations of zVAD.fmk, BV6, and human recombinant TNFα. Cell death was assessed by fluorescence‐based microscopic quantification of the fraction of PI‐positive cells, compared to total cells, using Hoechst 33342 and PI double staining (Sigma‐Aldrich). Imaging and quantification were performed using the ImageXpress Micro XLS Widefield High‐Content Analysis System and MetaXpress Software according to the manufacturer's instructions (Molecular Devices Sunnyvale, CA, USA).
Hoechst 33342 and pi double staining
Manufactured by Merck Group
Sourced in United States
Hoechst 33342 and PI double staining is a laboratory technique used to stain and identify different cell populations. Hoechst 33342 is a fluorescent dye that binds to DNA, allowing the visualization of nuclei. Propidium iodide (PI) is another fluorescent dye that can only enter cells with compromised membranes, such as dead or dying cells. The combination of these two dyes provides information about the viability and DNA content of cells in a sample.
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4 protocols using hoechst 33342 and pi double staining
1
Quantifying Cell Death Dynamics
2
Cell Viability and Death Assays
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Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Cell death was measured by flow cytometric analysis (FACS Canto II, BD Biosciences, Heidelberg, Germany) of DNA fragmentation of PI-stained nuclei as described before43 (link) or by fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (both Sigma-Aldrich) and ImageXpress Micro XLS Widefield High-Content Analysis System and MetaXpress Software according to the manufacturer's instructions (Molecular Devices Sunnyvale, CA, USA).
3
Fluorescence-Based Cell Death Assay
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Cell death was measured by fluorescence-based microscope analysis of PI uptake using Hoechst 33342 and PI double staining (Sigma-Aldrich, St. Louis, Missouri, USA) and the ImageXpress Micro XLS Widefield High-Content Analysis System and MetaXpress Software according to the manufacturer’s instructions (Molecular Devices Sunnyvale, CA, USA).
4
Autophagy Induction Screening in MZ-54 and ATG5 KO Cells
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Parental MZ-54 cells as well as ATG5 KO cells were seeded at 6500 cells/96-well followed by treatment with autophagy-inducing compounds of the Enzo Screen-Well™ library (Enzo Life Sciences, Lausen, Switzerland). Compounds were added to final concentrations between 100 nM and 100 µM. Cell death was assessed after 48 h by fluorescence-based microscope analysis of propidium iodide (PI) uptake using Hoechst 33342 and PI double staining (Sigma-Aldrich, St. Louis, Missouri, USA) as well as ImageXpress Micro XLS Widefield High-Content Analysis System and MetaXpress Software according to the manufacturer’s instructions (Molecular Devices Sunnyvale, CA, USA).
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