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Cellular senescence is a molecular process that is activated in response to a large variety of distinct stress signals. Mechanistically, cellular senescence is characterized by an arrest in cell cycle accompanied by phenotypic adaptations and physiological alterations including changes in the secretory profile of senescent cells termed the senescence-associated secretory phenotype (SASP). Here we describe a detailed, automation- compatible method for the detection of senescence-associated beta galactosidase (SA-β-gal) activity as a hallmark of cellular senescence using a conventional fluorescent microscope equipped with a transmitted light module. Moreover, we outline a protocol for the automated analysis of cellular senescence using convolutional neural networks (CNNs) and mathematical morphology. In sum, we provide a toolset for the high throughput assessment of cellular senescence based on light microscopy and automated image analysis., Competing Interests: Conflicts of interest OK is a cofounder of Samsara Therapeutics. GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Tollys, and Vascage. GK has been consulting for Reithera. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK's wife, Laurence Zitvogel, has held research contracts with 9 Meters Biopharma, Daiichi Sankyo, Pilege, was on the on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. GK's brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Anticancer drugs that suppress DNA-to-RNA transcription are particularly efficient in stimulating immunogenic cell death and hence eradicate malignant cells in a way that they will ignite an antitumor immune response. This is therapeutically relevant as it allows treatment response to last beyond drug discontinuation. For this reason, it is important to measure transcription inhibition in a precise fashion. Here, we detail two complementary assays for the assessment of transcription inhibition, one that detects the physical separation of fibrillarin and nucleolin by two-color immunofluorescence and another that measures the diminution of incorporated 5-ethynyl uridine (EU) into RNA, as revealed by click chemistry and the per-cell-intensity of a fluorescent signal., Competing Interests: Conflict of interest G.K. and O.K. are cofounders of Samsara Therapeutics. G.K. is a founder of everImmune and Therafast Bio., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Robotized high throughput screening allows for the assessment of autophagy in a large number of samples. Here, we describe a drug discovery platform for the phenotypic identification of novel autophagy inducers by means of automated cell biology workflows employing robotized cell culture, sample preparation and data acquisition. In this setting, fluorescent biosensor cells that express microtubule-associated proteins 1A/1B light chain 3B (best known as LC3) conjugated to green fluorescent protein (GFP), are utilized together with automated high content microscopy for the image-based assessment of autophagy. In sum, we detail a drug discovery screening workflow from high throughput sample preparation and processing to data acquisition and analysis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Macroautophagy (hereafter referred to as autophagy) serves the liberation of energy resources through the degradation of cellular components and is characterized by the formation of double-membraned vesicles, commonly referred to as autophagosomes. Microtubule-associated proteins 1A/1B light chain 3B (hereafter referred to as LC3) plays a crucial role during autophagosome formation, as cleavage of its immature form and subsequent conjugation to phosphatidylethanolamine facilitates autophagosomal membrane biogenesis. Indeed, the redistribution of green fluorescent protein (GFP)-conjugated LC3 from a diffuse cytosolic pattern into forming autophagosomes constitutes a morphological phenotype (commonly referred to as LC3 puncta) applicable to phenotypic analysis. The quantification of LC3 puncta in end-point assays has extensively been used in the past, allowing for the identification of autophagy modulators. Here, we describe a robust method employing automated confocal live cell imaging for the study of time-resolved LC3 dynamics. Furthermore, this method can be used to differentiate between phenotypes such as the homogeneous distribution of LC3 puncta in the cytoplasm, and the aggregation of LC3 clusters juxtaposed to the nucleus thus allowing for functional predictions., Competing Interests: Conflict of interest G.K. and O.K. are cofounders of Samsara Therapeutics. G.K. is a founder of everImmune and Therafast Bio., (Copyright © 2021 Elsevier Inc. All rights reserved.)