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In our earlier work, we presented Cellytics, a device that uses lens-free shadow imaging technology (LSIT) to monitor natural killer cell activity. Here, we present an improved Cellytics system that has been upgraded to a four-channel configuration to achieve higher throughput while maintaining robust reproducibility for rapid and cost-effective leukocyte analysis. The performance of this multi-channel Cellytics system was improved through refinements to the micro-pinhole chip. Etched pinholes provided better image resolution and clarity compared to drilled pinholes. To stimulate leukocytes, we used an activation stimulator cocktail (ASC) and quantified the resulting morphological changes using shadow-based metrics, including peak-to-peak distance (PPD) and maxima-to-minima standard deviation (MMD-SD). In addition, we developed a new leukocyte activation parameter (LAP) to specifically assess these activation-induced morphological changes. After ASC stimulation, leukocytes showed significantly increased PPD and LAP values and decreased MMD-SD compared to non-activated leukocytes. These results are consistent with the results of the flow cytometric analysis. These results emphasize the potential of Cellytics for the rapid and accurate assessment of leukocyte activation and provide a valuable tool for both clinical diagnostics and basic immunological research.<\/jats:p>","DOI":"10.3390\/bios15030143","type":"journal-article","created":{"date-parts":[[2025,2,24]],"date-time":"2025-02-24T10:04:28Z","timestamp":1740391468000},"page":"143","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Multi-Channel Cellytics for Rapid and Cost-Effective Monitoring of Leukocyte Activation"],"prefix":"10.3390","volume":"15","author":[{"given":"Hojin","family":"Cheon","sequence":"first","affiliation":[{"name":"Department of Electronics and Information Engineering, Korea University, Sejong 30019, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6302-6326","authenticated-orcid":false,"given":"Samir","family":"Kumar","sequence":"additional","affiliation":[{"name":"Department of Electronics and Information Engineering, Korea University, Sejong 30019, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inha","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Bioinformatics, Korea University, Sejong 30019, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7684-6043","authenticated-orcid":false,"given":"Sanghoon","family":"Shin","sequence":"additional","affiliation":[{"name":"Department of Electronics and Information Engineering, Korea University, Sejong 30019, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyeji","family":"Jang","sequence":"additional","affiliation":[{"name":"Department of Electronics and Information Engineering, Korea University, Sejong 30019, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6396-0859","authenticated-orcid":false,"given":"Young-Sun","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Hepatology, Guro Hospital, Korea University College of Medicine, Seoul 08308, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8737-6902","authenticated-orcid":false,"given":"Myung-Hyun","family":"Nam","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Anam Hospital, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyun Sik","family":"Jun","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Bioinformatics, Korea University, Sejong 30019, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7990-7390","authenticated-orcid":false,"given":"Sungkyu","family":"Seo","sequence":"additional","affiliation":[{"name":"Department of Electronics and Information Engineering, Korea University, Sejong 30019, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1111\/bjh.17022","article-title":"The Diagnostic Utility of Eosinophil Morphology","volume":"191","author":"Leach","year":"2020","journal-title":"Br. 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