{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"Research Square"}],"indexed":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T06:22:19Z","timestamp":1747203739371,"version":"3.40.5"},"posted":{"date-parts":[[2021,4,15]]},"group-title":"Protocol Exchange","reference-count":0,"publisher":"Springer Science and Business Media LLC","license":[{"start":{"date-parts":[[2021,4,15]],"date-time":"2021-04-15T00:00:00Z","timestamp":1618444800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2021,3,29]]},"abstract":"<title>Abstract<\/title>\n        <p>Toll-like receptors (TLRs) are part of the family of pattern recognition receptors (PRRs) that evolved to recognize pathogen-associated molecular patterns (PAMPs). A variety of PAMPs have been identified, from various microbial components to viral molecules. <italic>E.coli<\/italic> (<italic>Escherichia coli<\/italic>) has been widely used as a prototype Gram-negative bacteria with the capacity to stimulate TLRs, in particular to elicit TLR4-dependent immune responses. This receptor mainly recognizes an essential component of the outer membrane of Gram-negative bacteria: lipopolysaccharide (LPS). In vitro stimulation of immune cells for cytokine production can be achieved with the use of commercially-available purified TLR agonists, including LPS. Here we show how to prepare an alternative stimulator of TLR4 in a very fast an inexpensive way, paraformaldehyde-fixed preparations of <italic>E. coli. <\/italic>This reagent is a powerful tool for the <italic>in vitro <\/italic>activation of TLR4 that can be quantified and therefore used in defined amounts. Moreover, it is a safe reagent (the <italic>E.coli<\/italic> cells are fixed) and it is easy to prepare in large quantities and to store. Other microorganisms such as yeasts can also be fixed in this way.<\/p>","DOI":"10.21203\/rs.3.pex-1437\/v1","type":"posted-content","created":{"date-parts":[[2021,4,15]],"date-time":"2021-04-15T12:47:24Z","timestamp":1618490844000},"source":"Crossref","is-referenced-by-count":0,"title":["Fixation of microorganisms with paraformaldehyde for TLR stimulation."],"prefix":"10.21203","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6506-7829","authenticated-orcid":false,"given":"Ana","family":"Neves-Costa","sequence":"first","affiliation":[{"name":"Instituto Gulbenkian de Ciencia"}]}],"member":"297","container-title":[],"original-title":[],"link":[{"URL":"https:\/\/protocolexchange.researchsquare.com\/article\/pex-1437\/v1","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/protocolexchange.researchsquare.com\/article\/pex-1437\/v1.html","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,2]],"date-time":"2025-05-02T11:55:22Z","timestamp":1746186922000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.protocols.io\/view\/fixation-of-microorganisms-with-paraformaldehyde-f-kqdg3qzoev25\/v1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,15]]},"references-count":0,"URL":"https:\/\/doi.org\/10.21203\/rs.3.pex-1437\/v1","relation":{},"subject":[],"published":{"date-parts":[[2021,4,15]]},"subtype":"preprint"}}