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. 2021 Jan 14;2(1):100286.
doi: 10.1016/j.xpro.2020.100286. eCollection 2021 Mar 19.

Isolation of exophers from cardiomyocyte-reporter mouse strains by fluorescence-activated cell sorting

José Ángel Nicolás-Ávila  1 , María Sánchez-Diaz  1 , Andrés Hidalgo  1
Affiliations

Isolation of exophers from cardiomyocyte-reporter mouse strains by fluorescence-activated cell sorting

José Ángel Nicolás-Ávila et al. STAR Protoc. .

Abstract

Cardiac exophers are membrane-bound extracellular vesicles released by cardiomyocytes with varied content and an average diameter of 3.5 μm. Here, we provide a detailed protocol to enable the identification and purification of cardiomyocyte-derived exophers by using fluorescence-activated cell sorting for downstream cellular and molecular analysis. This protocol requires the use of mouse strains expressing fluorescent proteins in cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Nicolás-Ávila et al. (2020).

Keywords: Cell isolation; Flow cytometry/mass cytometry; Immunology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Perfusion and heart isolation for exopher preparation (A) Material needed: (1) cold PBS, (2) forceps, (3) surgery scissors, (4) dissection table, (5) 10 mL syringe with 21G needle loaded with cold PBS and (6) 70% ethanol. (B) Immobilization of the mouse. (C) First incision. (D) Skin removal. (E) Access to peritoneal cavity. (F) Access to thoracic cavity. Yellow arrows indicate the point where you should cut to separate the thorax and expose the heart. (G) Heart exposure. (H) Thorax immobilization. (I) Right atrium puncturing. (Left) Yellow arrow points to the right atrium. (Right) Once punctured, blood will start flowing out fast. (J) Needle insertion in left ventricle for perfusion. (K) Heart collection. You should cut below the forceps, as indicated by the yellow arrow. (L) (Left) Place the heart in a Petri dish with PBS. (Right) Remove the atria and big vessels (indicated by the yellow arrow). (M) Place the ventricular myocardium into a Falcon tube with fresh PBS. (N) Cardiac tissue before (left) and after (right) mincing with scissors in digestion buffer. (O) Transference of digested tissue through a 40 μm strainer placed in a 50 mL Falcon tube. (P) Summary of serial centrifugation steps.
Figure 2
Figure 2
Identification of cardiac exophers by flow cytometry (A and B) Gating strategy used to identify cardiac exophers based on (A) size, markers and (B) fluorescence in a cardiomyocyte-reporter mouse model (αMHCCRE Rosa26TdTom, referred as CardRED) (Nicolás-Ávila et al., 2020). (C) Back-gating of cardiac exophers, as identified in (A) and (B). (D) Bright-field images of sorted exophers.
See this image and copyright information in PMC

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