IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2.
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| Title: | IF-CRIB: A 3D-printable device to facilitate immunofluorescence experiments and its application in screening and characterizing cells expressing a degradable form of ERK2. |
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| Authors: | Berliocchi E; Université Côte d'Azur (UniCA), CNRS UMR 7284 and INSERM U 1081, Institute for Research on Cancer and Aging of Nice (IRCAN), 28 Avenue de Valombrose, 06107 Nice, France; Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, Quebec, Canada., Onesto C; Université Côte d'Azur (UniCA), CNRS UMR 7284 and INSERM U 1081, Institute for Research on Cancer and Aging of Nice (IRCAN), 28 Avenue de Valombrose, 06107 Nice, France; Polytech Nice Sophia, Bioengineering Department, Sophia Antipolis, France., Pagès G; Université Côte d'Azur (UniCA), CNRS UMR 7284 and INSERM U 1081, Institute for Research on Cancer and Aging of Nice (IRCAN), 28 Avenue de Valombrose, 06107 Nice, France., Lenormand P; Université Côte d'Azur (UniCA), CNRS UMR 7284 and INSERM U 1081, Institute for Research on Cancer and Aging of Nice (IRCAN), 28 Avenue de Valombrose, 06107 Nice, France., Buscà R; Université Côte d'Azur (UniCA), CNRS UMR 7284 and INSERM U 1081, Institute for Research on Cancer and Aging of Nice (IRCAN), 28 Avenue de Valombrose, 06107 Nice, France. Electronic address: roser.busca@univ-cotedazur.fr. |
| Source: | Methods (San Diego, Calif.) [Methods] 2025 Nov; Vol. 243, pp. 1-15. Date of Electronic Publication: 2025 Aug 09. |
| Publication Type: | Journal Article |
| Language: | English |
| Journal Info: | Publisher: Academic Press Country of Publication: United States NLM ID: 9426302 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1095-9130 (Electronic) Linking ISSN: 10462023 NLM ISO Abbreviation: Methods Subsets: MEDLINE |
| Imprint Name(s): | Publication: Duluth, MN : Academic Press Original Publication: San Diego : Academic Press, c1990- |
| MeSH Terms: | Mitogen-Activated Protein Kinase 1*/genetics , Mitogen-Activated Protein Kinase 1*/metabolism , Printing, Three-Dimensional*/instrumentation , Fluorescent Antibody Technique*/methods , Fluorescent Antibody Technique*/instrumentation, Animals ; Mice ; NIH 3T3 Cells ; Humans |
| Abstract: | Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Immunofluorescence-based detection of proteins in fixed cells is a powerful tool for research in cell and developmental biology. While a variety of immunofluorescence protocols exist, they can be time consuming or require expensive equipment which may not be accessible to all laboratories. A common challenge in these protocols is the numerous washing steps, particularly in experiments with numerous conditions. To address this, here we introduce the IF-CRIB device, a 3D-printable wash rack specifically designed for applications involving a high number of round coverslips with adherent cultured cells. We detail its design and the 3D printing process which can be easily used by any laboratory and we highlight that it facilitates the numerous washing steps. In addition, we present the IF-Express protocol, an optimized and effective method that enables fast and consistent immunofluorescence results. As an example of the utility of the IF-CRIB device and the IF-Express protocol, we describe their application in the screening and characterization of several NIH3T3 cell clones expressing a degradable form of ERK2 kinase (ERK2-dTAG) after treatment with the dTAG-13 compound. The generation of ERK2-dTAG clones involves a knock-in strategy. We provide a detailed methodology for clone selection, immunofluorescence screening, and characterization of ERK2-dTAG, including degradation kinetics, dose-response analysis, and nuclear translocation assays to assess ERK2-dTAG functionality. The IF-CRIB device and IF-Express protocol has been proven to be efficient for the obtention and characterization of ERK2dTAG-expressing clones thereby offering a powerful framework for studying ERK2 dynamics in cell biology and disease models. (Crown Copyright © 2025. Published by Elsevier Inc. All rights reserved.) |
| Contributed Indexing: | Keywords: 3D-printing; ERK quantity; ERK2; FKPB12(F36V); IF-CRIB; IF-Express protocol; Immunofluorescence; PROTAC; dTAG; dTAG-13 |
| Substance Nomenclature: | EC 2.7.11.24 (Mitogen-Activated Protein Kinase 1) |
| Entry Date(s): | Date Created: 20250811 Date Completed: 20250916 Latest Revision: 20250916 |
| Update Code: | 20250916 |
| DOI: | 10.1016/j.ymeth.2025.08.004 |
| PMID: | 40789436 |
| Database: | MEDLINE |
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Nájsť tento článok vo Web of Science | Abstract: | Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.<br />Immunofluorescence-based detection of proteins in fixed cells is a powerful tool for research in cell and developmental biology. While a variety of immunofluorescence protocols exist, they can be time consuming or require expensive equipment which may not be accessible to all laboratories. A common challenge in these protocols is the numerous washing steps, particularly in experiments with numerous conditions. To address this, here we introduce the IF-CRIB device, a 3D-printable wash rack specifically designed for applications involving a high number of round coverslips with adherent cultured cells. We detail its design and the 3D printing process which can be easily used by any laboratory and we highlight that it facilitates the numerous washing steps. In addition, we present the IF-Express protocol, an optimized and effective method that enables fast and consistent immunofluorescence results. As an example of the utility of the IF-CRIB device and the IF-Express protocol, we describe their application in the screening and characterization of several NIH3T3 cell clones expressing a degradable form of ERK2 kinase (ERK2-dTAG) after treatment with the dTAG-13 compound. The generation of ERK2-dTAG clones involves a knock-in strategy. We provide a detailed methodology for clone selection, immunofluorescence screening, and characterization of ERK2-dTAG, including degradation kinetics, dose-response analysis, and nuclear translocation assays to assess ERK2-dTAG functionality. The IF-CRIB device and IF-Express protocol has been proven to be efficient for the obtention and characterization of ERK2dTAG-expressing clones thereby offering a powerful framework for studying ERK2 dynamics in cell biology and disease models.<br /> (Crown Copyright © 2025. Published by Elsevier Inc. All rights reserved.) |
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| ISSN: | 1095-9130 |
| DOI: | 10.1016/j.ymeth.2025.08.004 |