c3d96dc88e66276aab3cde4a51b0dba8 life-15-00410.pdf 88ca3a2a58c56a0fcead248915896007780bcf51 life-15-00410.pdf 700a11958953bb859073f828da13f27fe2440ec854d58fdecf673cb7fdab691c life-15-00410.pdf Title: Comparison of Methods for Isolating Exosomes from Plasma Subjects with Normal and High Fat Percentages "2279 Subject: Adipose tissue is responsible for fat storage and is an important producer of extracellular vesicles (EVs). The biological content of exosomes, one kind of EV, provides information on aspects such as immunometabolic alterations. This study aimed to compare three plasma exosome isolation methods—using a commercial kit (CK), size exclusion chromatography (SEC), and differential centrifugation (DC)—and select the best one. Individuals categorized by normal and high body fat percentages were used. The DC and CK were proven to be the most advantageous out of the exosome isolation methods, so we suggest these methods for further protein and molecular analyses, respectively. Still, we emphasize the importance of selecting an appropriate methodology depending on the specific research objectives. At the same time, no statistical differences in exosome quality, morphology, total protein, or microRNA concentration were observed between individuals categorized by body fat percentage, so we suggest that the exosomal cargo varies in individuals with normal and high fat percentages. Keywords: plasma exosomes; exosome isolation methods; fat percentage Author: Jacqueline Noboa-Velástegui, Juan Carlos León, Jorge Castro, Ana Fletes, Perla Madrigal, Iñaki Álvarez and Rosa Navarro Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu Mar 6 18:01:25 2025 CET ModDate: Thu Mar 6 18:05:31 2025 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 11 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1283540 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- FPVFVL+URWPalladioL-Roma Type 1 Custom yes yes yes 10 0 LYMILD+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 XCZREK+URWPalladioL-Ital Type 1 Custom yes yes yes 21 0 ZXDKIP+EURM10 Type 1 Builtin yes yes yes 47 0 ITYRUV+VnURWPalladioL-Italic Type 1 Custom yes yes yes 55 0 VYVZGZ+CMSY10 Type 1 Builtin yes yes yes 61 0 BCDEEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 77 0 BCDFEE+MICROSOFT SANS SERIF TrueType WinAnsi yes yes no 86 0 Arial TrueType WinAnsi no no no 90 0 Arial,Italic TrueType WinAnsi no no no 94 0 Symbol CID TrueType Identity-H yes no yes 97 0 EGHKPB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 120 0 EGHKPC+PalatinoLinotype CID TrueType Identity-H yes yes yes 123 0 EGHKPD+PalatinoLinotype TrueType WinAnsi yes yes no 129 0 EGHLAD+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 132 0 EGHLAE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 138 0 EGHLEE+PalatinoLinotype TrueType MacRoman yes yes no 141 0 EGHKPB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 155 0 EGHKPC+PalatinoLinotype CID TrueType Identity-H yes yes yes 158 0 EGHKPD+PalatinoLinotype TrueType WinAnsi yes yes no 164 0 EGHLAD+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 167 0 EGHLAE+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 173 0 EGHLEE+PalatinoLinotype TrueType MacRoman yes yes no 176 0 NROZPH+VnURWPalladioL Type 1 Custom yes yes yes 190 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes yes 195 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-04-10 02:08:00 CEST RepresentationInformation: life-15-00410.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-04-09 14:41:51 CEST Size: 1283540 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 366 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Samples Destination: subsection.2.1 Item: Title: Exosome Isolation and Characterization Destination: subsection.2.2 Children: Item: Title: Isolation by Differential Centrifugation (DC) Destination: subsubsection.2.2.1 Item: Title: Isolation by Size Exclusion Chromatography (SEC) Destination: subsubsection.2.2.2 Item: Title: Isolation by Precipitation with a Commercial Kit (CK) Destination: subsubsection.2.2.3 Item: Title: Characterization by Dynamic Light Scattering (DLS) Destination: subsubsection.2.2.4 Item: Title: Characterization by Cryo-TEM and TEM Destination: subsubsection.2.2.5 Item: Title: Characterization by Western Blot Destination: subsubsection.2.2.6 Item: Title: MicroRNA Isolation Destination: subsection.2.3 Item: Title: Statistical and Image Analyses Destination: subsection.2.4 Item: Title: Results Destination: section.3 Children: Item: Title: The Exosome Isolation Methods Showed Equal Performances in Total Protein and microRNA Concentration, While an Inverse Pattern Was Observed Among Individuals with High Fat Mass Contents Destination: subsection.3.1 Item: Title: The Morphology and Quality of the Three Exosome Isolation Methods Were as Expected, with Inconsistencies in Purity Destination: subsection.3.2 Item: Title: CD9 and CD81 Do Not Differ Between Normal- and High-Fat-Percentage Individuals in SEC Fractions Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: Appendix A Destination: appendix.A. Item: Title: References Destination: appendix.B. Info: Title: Comparison of Methods for Isolating Exosomes from Plasma Subjects with Normal and High Fat Percentages "2279 Author: Jacqueline Noboa-Velástegui, Juan Carlos León, Jorge Castro, Ana Fletes, Perla Madrigal, Iñaki Álvarez and Rosa Navarro Subject: Adipose tissue is responsible for fat storage and is an important producer of extracellular vesicles (EVs). The biological content of exosomes, one kind of EV, provides information on aspects such as immunometabolic alterations. This study aimed to compare three plasma exosome isolation methods using a commercial kit (CK), size exclusion chromatography (SEC), and differential centrifugation (DC) and select the best one. Individuals categorized by normal and high body fat percentages were used. The DC and CK were proven to be the most advantageous out of the exosome isolation methods, so we suggest these methods for further protein and molecular analyses, respectively. Still, we emphasize the importance of selecting an appropriate methodology depending on the specific research objectives. At the same time, no statistical differences in exosome quality, morphology, total protein, or microRNA concentration were observed between individuals categorized by body fat percentage, so we suggest that the exosomal cargo varies in individuals with normal and high fat percentages. 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