{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T05:06:10Z","timestamp":1790053570847,"version":"4.0.1"},"reference-count":26,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T00:00:00Z","timestamp":1789776000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Education and Science of the Russian Federation","award":["075-15-2025-464"],"award-info":[{"award-number":["075-15-2025-464"]}]},{"award":["075-15-2025-464"],"award-info":[{"award-number":["075-15-2025-464"]}],"id":[{"id":"https:\/\/ror.org\/00ghqgy32","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Methods Protoc.","MPs"],"abstract":"<jats:p>Isolation of pancreatic islets for single-cell RNA sequencing (scRNA-seq) remains technically challenging because enzymatic digestion, mechanical dissociation, and subsequent purification can cause cell loss, aggregation, and processing-induced transcriptional changes. This study aimed to adapt a practical gradient-free alternative for preparing murine pancreatic islets for downstream single-cell transcriptomic analysis while reducing the number of sample-processing steps. The workflow combined intraductal perfusion of the pancreas with 3 mL of a collagenase solution through the common bile duct, controlled enzymatic digestion at 37 \u00b0C, performed manual islet selection without density-gradient purification, and achieved gentle dissociation with Accutase and EDTA. DNase I was included in all post-digestion steps to limit cell aggregation. The protocol was evaluated using pancreatic islets from BKS.Cg-Dock7m +\/+ Leprdb\/J mice, a model of type 2 diabetes. Compared with Ficoll-based purification, the gradient-free protocol provided viable cell yields within a similar range while eliminating density-gradient preparation, an 18 min gradient centrifugation step, two subsequent wash centrifugations, and the associated transfer and dilution steps. The resulting scRNA-seq library showed satisfactory quality-control (QC) characteristics and retained the major endocrine populations, including \u03b2-, \u03b1-, \u03b4-, and PP cells. The gradient-free sample also showed a lower overall stress-response score and lower expression of several immediate early genes, although heat shock- and endoplasmic reticulum (ER) stress-associated genes displayed heterogeneous patterns. These differences were considered descriptive because only one pooled scRNA-seq library was generated for each workflow. These findings support gradient-free isolation as a possible alternative to Ficoll-based purification for preparing murine pancreatic islets for scRNA-seq analysis.<\/jats:p>","DOI":"10.3390\/mps9050136","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T09:01:38Z","timestamp":1789981298000},"page":"136","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Gradient-Free Isolation of Murine Pancreatic Islets for Single-Cell RNA Sequencing"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1361-666X","authenticated-orcid":false,"given":"Evgeny S.","family":"Ruchko","sequence":"first","affiliation":[{"name":"Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Zakhar R.","family":"Starinnov","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marat S.","family":"Sabirov","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria B.","family":"Chernysheva","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3341-4248","authenticated-orcid":false,"given":"Arthur A.","family":"Lee","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vagif Ali oglu","family":"Gasanov","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"given":"Andrey V.","family":"Vasiliev","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, 119334 Moscow, Russia"},{"name":"Department of Embryology, Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1016\/j.ajt.2024.02.011","article-title":"Inflammatory and Hypoxic Stress-Induced Islet Exosomes Released during Isolation Are Associated with Poor Transplant Outcomes in Islet Autotransplantation","volume":"24","author":"Saravanan","year":"2024","journal-title":"Am. 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