Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method
Accurate identification of cell cycle stages is essential for investigating fundamental biological processes such as proliferation, differentiation, and tumorigenesis. While flow cytometry remains a widely used technique for such analyses, it is limited by its lack of single-cell resolution and its...
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Bio-protocol LLC
2025-08-01
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| author | Syon Reddy Yu-Lin Chen Aussie Suzuki |
| author_facet | Syon Reddy Yu-Lin Chen Aussie Suzuki |
| author_sort | Syon Reddy |
| collection | DOAJ |
| description | Accurate identification of cell cycle stages is essential for investigating fundamental biological processes such as proliferation, differentiation, and tumorigenesis. While flow cytometry remains a widely used technique for such analyses, it is limited by its lack of single-cell resolution and its requirement for large sample sizes due to its population-based approach. These limitations underscore the need for alternative or complementary methods that offer single-cell precision with compatibility for small-scale applications. We present ImmunoCellCycle-ID, an immunofluorescence-based method that leverages the spatial distribution of endogenous markers, such as DNA, proliferating cell nuclear antigen (PCNA), centromere protein F (CENP-F), and centromere protein C (CENP-C), to reliably distinguish G1, early S, late S, early G2, late G2, and all mitotic sub-stages. This technique does not rely on precise signal quantification and utilizes standard immunofluorescence protocols alongside conventional laboratory microscopes, ensuring broad accessibility. Importantly, ImmunoCellCycle-ID detects endogenous proteins without the need for genetic modification, making it readily applicable to a wide range of human cell lines. Beyond its utility for single-cell resolution, the method can be scaled for population-level analyses, similar to flow cytometry. With its precision, versatility, and ease of implementation, ImmunoCellCycle-ID offers a powerful tool for high-resolution cell cycle profiling across diverse experimental platforms. |
| format | Article |
| id | doaj-art-a3befd44874343bfa3a409cc4e152d67 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Bio-protocol LLC |
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| series | Bio-Protocol |
| spelling | doaj-art-a3befd44874343bfa3a409cc4e152d672025-08-20T04:01:57ZengBio-protocol LLCBio-Protocol2331-83252025-08-01151510.21769/BioProtoc.5407Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID MethodSyon Reddy0Yu-Lin Chen1Aussie Suzuki2McArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, Madison, WI, USAMcArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, Madison, WI, USAMcArdle Laboratory for Cancer Research, Department of Oncology, University of Wisconsin-Madison, Madison, WI, USACarbone Cancer Center, University of Wisconsin-Madison, Madison, WI, USAAccurate identification of cell cycle stages is essential for investigating fundamental biological processes such as proliferation, differentiation, and tumorigenesis. While flow cytometry remains a widely used technique for such analyses, it is limited by its lack of single-cell resolution and its requirement for large sample sizes due to its population-based approach. These limitations underscore the need for alternative or complementary methods that offer single-cell precision with compatibility for small-scale applications. We present ImmunoCellCycle-ID, an immunofluorescence-based method that leverages the spatial distribution of endogenous markers, such as DNA, proliferating cell nuclear antigen (PCNA), centromere protein F (CENP-F), and centromere protein C (CENP-C), to reliably distinguish G1, early S, late S, early G2, late G2, and all mitotic sub-stages. This technique does not rely on precise signal quantification and utilizes standard immunofluorescence protocols alongside conventional laboratory microscopes, ensuring broad accessibility. Importantly, ImmunoCellCycle-ID detects endogenous proteins without the need for genetic modification, making it readily applicable to a wide range of human cell lines. Beyond its utility for single-cell resolution, the method can be scaled for population-level analyses, similar to flow cytometry. With its precision, versatility, and ease of implementation, ImmunoCellCycle-ID offers a powerful tool for high-resolution cell cycle profiling across diverse experimental platforms.https://bio-protocol.org/en/bpdetail?id=5407&type=0 |
| spellingShingle | Syon Reddy Yu-Lin Chen Aussie Suzuki Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method Bio-Protocol |
| title | Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method |
| title_full | Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method |
| title_fullStr | Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method |
| title_full_unstemmed | Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method |
| title_short | Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method |
| title_sort | accurate identification of cell cycle stages in rpe1 cells using the immunocellcycle id method |
| url | https://bio-protocol.org/en/bpdetail?id=5407&type=0 |
| work_keys_str_mv | AT syonreddy accurateidentificationofcellcyclestagesinrpe1cellsusingtheimmunocellcycleidmethod AT yulinchen accurateidentificationofcellcyclestagesinrpe1cellsusingtheimmunocellcycleidmethod AT aussiesuzuki accurateidentificationofcellcyclestagesinrpe1cellsusingtheimmunocellcycleidmethod |