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J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Chromosome preparation from peripheral blood in felids is often difficult because lymphocytes respond inconsistently to standard mitogens like phytohemagglutinin (PHA), resulting in low and variable mitotic indices. Here, we present a standardized and reproducible protocol for lymphocyte culture and metaphase chromosome preparation in felids using concanavalin A (ConA) as a mitogenic stimulus. A total of 300 \u00b5L of peripheral blood consistently produced optimal chromosome preparations and was cultured in RPMI 1640 medium supplemented with fetal bovine serum, antibiotics, and Concanavalin A (40 \u00b5g\/mL), then incubated at 37 \u00b0C for 70 h. Mitotic arrest was induced with colchicine, and cells were subjected to hypotonic treatment and fixation using methanol\u2013acetic acid before slide preparation and Giemsa staining. This approach consistently yielded high-quality metaphase spreads across the eight felid species examined, with mean values ranging from 32 \u00b1 5 to 55 \u00b1 7 metaphases per slide, while maintaining chromosome morphology appropriate for cytogenetic investigation. Compared with fibroblast-based approaches, this methodology is less invasive and more feasible for clinical and conservation applications because it relies on peripheral blood collection rather than tissue biopsy, reducing the invasiveness of sampling procedures. This optimized lymphocyte culture workflow enhances mitotic yield and repeatability, serving as a significant resource for cytogenetic research, particularly in veterinary genetics, evolutionary biology, and the conservation of endangered felid species.<\/jats:p>","DOI":"10.3390\/ijms27156662","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T09:47:41Z","timestamp":1785145661000},"page":"6662","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Concanavalin A Protocol for Felid Lymphocyte Culture and Chromosome Preparation"],"prefix":"10.3390","volume":"27","author":[{"given":"Pr\u00edncia Grejo","family":"Setti","sequence":"first","affiliation":[{"name":"Department of Genetics and Evolution, Federal University of S\u00e3o Carlos, S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alan Moura","family":"de Oliveira","sequence":"additional","affiliation":[{"name":"Department of Genetics and Evolution, Federal University of S\u00e3o Carlos, S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"St\u00e9phanie Ferguson","family":"Motheo","sequence":"additional","affiliation":[{"name":"Central Paulista University Center, S\u00e3o Carlos 13563-470, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ot\u00e1vio Guilherme Gon\u00e7alves","family":"de Almeida","sequence":"additional","affiliation":[{"name":"Wildlife Screening and Rehabilitation Center (CETRAS), Morro de S\u00e3o Bento, Ribeir\u00e3o Preto 14091-902, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1672-3054","authenticated-orcid":false,"given":"Thomas","family":"Liehr","sequence":"additional","affiliation":[{"name":"Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, 07747 Jena, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4340-1464","authenticated-orcid":false,"given":"Marcelo de Bello","family":"Cioffi","sequence":"additional","affiliation":[{"name":"Department of Genetics and Evolution, Federal University of S\u00e3o Carlos, S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1292\/jvms.25-0265","article-title":"37,X\/38,XY mosaicism in a cat with a female phenotype and an enlarged clitoris","volume":"88","author":"Yoshimura","year":"2026","journal-title":"J. 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Am."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1590\/1678-4162-13078","article-title":"Comparative Cytogenetics in Felids (Carnivora: Felidae): Leopardus Wiedii, Panthera Onca, Puma Concolor and Felis Catus","volume":"76","author":"Gunski","year":"2024","journal-title":"Arq. Bras. Med. Vet. Zootec."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1093\/jhered\/esm130","article-title":"The Ancestral Carnivore Karyotype (2n = 38) Lives Today in Ringtails","volume":"99","author":"Nash","year":"2008","journal-title":"J. Hered."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1159\/000063032","article-title":"Comparative Molecular Cytogenetic Studies in the Order Carnivora: Mapping Chromosomal Rearrangements onto the Phylogenetic Tree","volume":"96","author":"Graphodatsky","year":"2002","journal-title":"Cytogenet. Genome Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1086\/282273","article-title":"Karyological Studies of Nine Species of Felidae","volume":"97","author":"Hsu","year":"1963","journal-title":"Am. Nat."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"55","DOI":"10.3409\/fb_73-2.06","article-title":"Comparative Cytogenetic Analysis of Selected Chromosomes in Felidae Family Members Using Zoo-FISH","volume":"73","author":"Cernohorska","year":"2025","journal-title":"Folia Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1038\/hdy.2011.107","article-title":"Chromosomal Rearrangements and Karyotype Evolution in Carnivores Revealed by Chromosome Painting","volume":"108","author":"Nie","year":"2012","journal-title":"Heredity"},{"key":"ref_14","first-page":"377","article-title":"Giemsa Banding Patterns in the Chromosomes of Twelve Species of Cats (Felidae)","volume":"12","author":"Gray","year":"1973","journal-title":"Cytogenet. Genome Res."},{"key":"ref_15","first-page":"306","article-title":"The Interrelationships of Chromosome Banding Patterns in Procyonids, Viverrids, and Felids","volume":"15","author":"Gray","year":"1975","journal-title":"Cytogenet. Genome Res."},{"key":"ref_16","first-page":"141","article-title":"Cytogenetic Analysis in Domestic Cats","volume":"47","author":"Ghotage","year":"2019","journal-title":"Int. J. Sci. Basic Appl. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1016\/0014-4827(60)90138-5","article-title":"Chromosome Preparations of Leukocytes Cultured from Human Peripheral Blood","volume":"20","author":"Moorhead","year":"1960","journal-title":"Exp. Cell Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1007\/s13237-016-0174-3","article-title":"An Optimized Protocol for Obtaining Mitotic Chromosomes from Cultured Reptilian Lymphocytes","volume":"59","author":"Viana","year":"2016","journal-title":"Nucleus"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1508\/cytologia.73.61","article-title":"Karyological study of the jungle cat, Felis chaus (Carnivora, Felidae), by conventional staining, G-banding, and high-resolution staining technique","volume":"73","author":"Tanomtong","year":"2008","journal-title":"Cytologia"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1717","DOI":"10.1073\/pnas.73.5.1717","article-title":"Effects of Concanavalin A and a Succinylated Derivative on Lymphocyte Proliferation and Cyclic Nucleotide Levels","volume":"73","author":"Hadden","year":"1976","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","first-page":"237","article-title":"The Use of Concanavalin A to Study the Immunoregulation of Human T Cells","volume":"46","author":"Dwyer","year":"1981","journal-title":"Clin. Exp. Immunol."},{"key":"ref_22","first-page":"226","article-title":"Characterization and Mitogenesis of Feline Lymphocyte Populations","volume":"68","author":"Rojko","year":"1982","journal-title":"Int. Arch. Allergy Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1969","DOI":"10.2460\/ajvr.1977.38.12.1969","article-title":"Responses of Enriched Populations of Feline T and B Lymphocytes to Mitogen Stimulation","volume":"38","author":"Taylor","year":"1977","journal-title":"Am. J. Vet. Res."},{"key":"ref_24","first-page":"148","article-title":"Generation of Concanavalin A-Induced Suppressor Cells in the Cat","volume":"69","author":"Langweiler","year":"1982","journal-title":"Int. Arch. Allergy Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Brooks, M.B., Harr, K.E., Seelig, D.M., Wardrop, K.J., and Weiss, D.J. (2022). Schalm\u2019s Veterinary Hematology, Wiley. [1st ed.].","DOI":"10.1002\/9781119500537"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1016\/j.lfs.2018.06.003","article-title":"Lectins as Mitosis Stimulating Factors: Briefly Reviewed","volume":"207","author":"Carvalho","year":"2018","journal-title":"Life Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1007\/s10540-005-2798-4","article-title":"An Improved Culture System of Mouse Peripheral Blood Lymphocytes for Analysis of Radiation-Induced Chromosome Aberrations","volume":"24","author":"Kanda","year":"2004","journal-title":"Biosci. 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