f8e42a5e3b9cb922bbff8e22212fac86 ijms-26-04951-v2.pdf 2a6c06ed0b3e7c5958383e8ad8ccc25468b44faf ijms-26-04951-v2.pdf 0a16b9733bc5ac8097ef4d914ded72b18ca7300e154a89ad5a533d734878fe02 ijms-26-04951-v2.pdf Title: Controlled Exit from the G2/M Checkpoint in RPE-1 Cells Using RO3306: Enrichment of Phase-Specific Cell Populations for In-Depth Analyses of Mitotic Events Subject: Studying the cell cycle is essential for understanding the molecular mechanisms that regulate cell division, growth, and differentiation in living organisms. However, mitosis constitutes only a brief phase of the overall cell cycle, making its analysis challenging in asynchronous cell populations due to its transient and dynamic nature. Cell synchronization methods help to enrich populations at specific cell cycle stages, including mitosis, typically by using chemical inhibitors to arrest cells at defined checkpoints. However, many existing protocols rely on combinations of inhibitors that interfere with normal mitotic progression, disrupting dynamics and causing side effects such as chromosome non-disjunction or lagging chromosomes, which limit their applicability. In this study, we present an RO3306 block-and-release strategy to selectively enrich cell populations at defined mitotic stages without compromising cell viability or disrupting their progression to mitotic exit. This approach provides a reliable method for studying mitotic events with high temporal resolution. Furthermore, by preserving mitotic integrity, it offers a valuable framework for investigating the molecular mechanisms of cell division and the processes driving genomic instability in human cells. Keywords: cell division; mitosis; RO3306; synchronization; cell cycle Author: Teresa Anglada, Núria Pulido-Artola, Marina Rodriguez-Muñoz and Anna Genesca Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu May 22 09:52:42 2025 CEST ModDate: Thu May 22 10:24:07 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3237538 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MIKRSI+VnURWPalladioL Type 1 Custom yes yes yes 10 0 PLEQSX+URWPalladioL-Roma Type 1 Custom yes yes yes 16 0 IIYXCK+URWPalladioL-Bold Type 1 Custom yes yes yes 22 0 PQYBDE+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 MDJLZU+EURM10 Type 1 Builtin yes yes yes 56 0 FDJNBD+PalatinoLinotype TrueType WinAnsi yes yes no 69 0 FDJNBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 86 0 FDJNBD+PalatinoLinotype TrueType WinAnsi yes yes no 89 0 FDJNBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 103 0 FDJNBD+PalatinoLinotype TrueType WinAnsi yes yes no 106 0 FDJNBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 123 0 FDJNBD+PalatinoLinotype TrueType WinAnsi yes yes no 126 0 FDJNBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 141 0 FDJNBD+PalatinoLinotype TrueType WinAnsi yes yes no 144 0 YFFKLH+CMSY10 Type 1 Builtin yes yes yes 160 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-06-12 02:07:54 CEST RepresentationInformation: ijms-26-04951-v2.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-06-11 12:49:38 CEST Size: 3237538 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 323 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Enrichment of Mitotic Cells Using an RO3306 Block-And-Release Method Destination: subsection.2.1 Item: Title: Controlled Enrichment of Mitotic Populations Destination: subsection.2.2 Children: Item: Title: Morphological Classification of Mitotic Cells Destination: subsubsection.2.2.1 Item: Title: Mitosis on Demand: Precise Timing for the Enrichment of Specific Mitotic Sub-Populations Destination: subsubsection.2.2.2 Item: Title: Applications of RO3306 Synchronization and Precise Washout Protocol Destination: subsection.2.3 Children: Item: Title: Enrichment of Metaphase Cells for Preparing Chromosome Spreads Destination: subsubsection.2.3.1 Item: Title: Enrichment of Cells at Mitotic Exit for Chromosomal Segregation Studies Destination: subsubsection.2.3.2 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Culture Destination: subsection.4.1 Item: Title: Lentiviral Particle Production and Transduction Destination: subsection.4.2 Item: Title: Cell Cycle Synchronization and Cell Treatments Destination: subsection.4.3 Item: Title: Cell Cycle Destination: subsection.4.4 Item: Title: Live/Dead Assay Destination: subsection.4.5 Item: Title: Immunofluorescence Destination: subsection.4.6 Item: Title: Fluorescence In Situ Hybridization Destination: subsection.4.7 Item: Title: Live-Cell Imaging Destination: subsection.4.8 Item: Title: Statistics Destination: subsection.4.9 Item: Title: References Destination: appendix.A. Info: Title: Controlled Exit from the G2/M Checkpoint in RPE-1 Cells Using RO3306: Enrichment of Phase-Specific Cell Populations for In-Depth Analyses of Mitotic Events Author: Teresa Anglada, Núria Pulido-Artola, Marina Rodriguez-Muñoz and Anna Genesca Subject: Studying the cell cycle is essential for understanding the molecular mechanisms that regulate cell division, growth, and differentiation in living organisms. However, mitosis constitutes only a brief phase of the overall cell cycle, making its analysis challenging in asynchronous cell populations due to its transient and dynamic nature. Cell synchronization methods help to enrich populations at specific cell cycle stages, including mitosis, typically by using chemical inhibitors to arrest cells at defined checkpoints. However, many existing protocols rely on combinations of inhibitors that interfere with normal mitotic progression, disrupting dynamics and causing side effects such as chromosome non-disjunction or lagging chromosomes, which limit their applicability. In this study, we present an RO3306 block-and-release strategy to selectively enrich cell populations at defined mitotic stages without compromising cell viability or disrupting their progression to mitotic exit. This approach provides a reliable method for studying mitotic events with high temporal resolution. Furthermore, by preserving mitotic integrity, it offers a valuable framework for investigating the molecular mechanisms of cell division and the processes driving genomic instability in human cells. Keywords: cell division; mitosis; RO3306; synchronization; cell cycle Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu May 22 09:52:42 CEST 2025 ModDate: Thu May 22 10:24:07 CEST 2025 ID: 0x436223bd7bdc3b3f96360945487f6dfe, 0x436223bd7bdc3b3f96360945487f6dfe Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: PLEQSX+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 248 FontDescriptor: FontName: PLEQSX+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: YFFKLH+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: YFFKLH+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true ToUnicode: true Font: BaseFont: IIYXCK+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 250 FontDescriptor: FontName: IIYXCK+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: 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