{
  "abstract": "Summary The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a step-by-step protocol for synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large circular unattached gels customizable in shape and size. We outline methods for their use in cell culture and downstream applications involving secretome or cell analysis, and protein visualization by fluorescence microscopy. This protocol is based on the recent work of Shi & Janmey who describe a novel and straightforward method for the production of large PAAm hydrogels for bulk cell culture and mechanobiology studies. 1 Their procedure results in one large gel that is not attached to a supporting surface and therefore can be transferred and/or stamped to generate PAAm gels of custom shapes and sizes. The aim of this step-by-step procedure is therefore not to improve the reported protocol, but to create a clearly outlined and repeatable protocol that enables a smooth implementation in any lab for a diverse audience. In addition, our protocol describes besides harvesting of cells also the collection of secretome for downstream biochemical analyses, as well as immunofluorescence labeling using antibodies that can readily be multiplexed for optimization of labeling conditions. Graphical abstract",
  "abstract_shown": true,
  "affiliations": [
    "Molecular Imaging and Photonics Division, Chemistry Department, Faculty of Sciences, KU Leuven, Celestijnenlaan 200F 3001 Heverlee, Belgium",
    "Molecular Materials group, Institute for Molecules and Materials, Faculty of Science, Radboud University, Heyendaalseweg 135, Nijmegen AJ 6525, the Netherlands",
    "Molecular Materials group, Institute for Molecules and Materials, Faculty of Science, Radboud University, Heyendaalseweg 135, Nijmegen AJ 6525, the Netherlands; Institute for Chemical Immunology, Nijmegen AJ 6525, the Netherlands",
    "Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, Madrid 28220, Spain",
    "Chronic Disease Programme, UFIEC, Instituto de Salud Carlos III, Madrid 28220, Spain; CIBER frailty and healthy aging (CIBERFES) 28029 Madrid, Spain",
    "Molecular Materials group, Institute for Molecules and Materials, Faculty of Science, Radboud University, Heyendaalseweg 135, Nijmegen AJ 6525, the Netherlands. Electronic address: p.kouwer@science.ru.nl",
    "Molecular Imaging and Photonics Division, Chemistry Department, Faculty of Sciences, KU Leuven, Celestijnenlaan 200F 3001 Heverlee, Belgium. Electronic address: susana.rocha@kuleuven.be"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 8,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Charlotte Cresens",
      "orcid": "0000-0001-8414-0913"
    },
    {
      "affs": [
        0
      ],
      "name": "Samet Aytekin",
      "orcid": "0000-0002-9369-9716"
    },
    {
      "affs": [
        1
      ],
      "name": "Behrad Shaghaghi",
      "orcid": "0000-0002-7259-6814"
    },
    {
      "affs": [
        2
      ],
      "name": "Lotte Gerrits",
      "orcid": "0009-0001-9594-5802"
    },
    {
      "affs": [
        3
      ],
      "name": "Ana Montero‐Calle",
      "orcid": "0000-0001-5141-0454"
    },
    {
      "affs": [
        4
      ],
      "name": "Rodrigo Barderas",
      "orcid": "0000-0003-3539-7469"
    },
    {
      "affs": [
        5
      ],
      "name": "Paul H. J. Kouwer",
      "orcid": "0000-0002-2760-191X"
    },
    {
      "affs": [
        6
      ],
      "name": "Susana Rocha",
      "orcid": "0000-0003-1258-9396"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/3qmefzdpvsnya.bib",
  "cited_by": 5,
  "doi": "10.1016/j.xpro.2026.104606",
  "fulltext_url": null,
  "id": "3qmefzdpvsnya",
  "issns": [
    "2666-1667"
  ],
  "landing_url": "https://doi.org/10.1016/j.xpro.2026.104606",
  "language": "en",
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "2502|Hydrogels: synthesis, properties, applications"
    ]
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  "openalex": "W7164003984",
  "pdf_url": null,
  "pmcid": "PMC13273567",
  "pmid": "42268723",
  "published": "2026-06-01",
  "publisher": "Elsevier BV",
  "ris": "https://research.draconitic.com/paper/3qmefzdpvsnya.ris",
  "sources": [
    {
      "first_seen": "2026-11-06T13:20:45Z",
      "native_id": "W7164003984",
      "source": "openalex",
      "url": "https://doi.org/10.1016/j.xpro.2026.104606"
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  "subjects": [
    {
      "field": "chemistry",
      "origin": "openalex:2502|Hydrogels: synthesis, properties, applications",
      "subfield": "materials-polymers"
    }
  ],
  "title": "Protocol for synthesis and mechanical characterization of polyacrylamide hydrogels of varying stiffness for cell culture applications",
  "title_text": "Protocol for synthesis and mechanical characterization of polyacrylamide hydrogels of varying stiffness for cell culture applications",
  "type": "peer-reviewed",
  "updated_at": "2026-11-06T13:20:45Z",
  "url": "https://research.draconitic.com/paper/3qmefzdpvsnya",
  "venue": "STAR Protocols",
  "versions": []
}