Key result
Optimized protocols using low-speed centrifugation, 40-μm filtering, and 1% paraformaldehyde fixation improved immune cell yield and preserved cellular morphology compared to standard methods.
Why the study?
Experimental approaches to isolate cardiac mononuclear cells are diverse, and detailed protocols enabling maximum live cell yields in the shortest time are not readily available.
This study provides an optimized, time-efficient protocol for isolating and characterizing immune cells from ischemic hearts, improving cell yield and preserving morphology for flow cytometry.
Enhances preclinical flow cytometry workflows for ischemic myocardium; leaves open whether these methodological refinements translate to novel therapeutic.
Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints. NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-μm filter can replace density-mediated mononuclear cell separation which usually results in 50–70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.
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Covarrubias et al. (2019) studied Ischemic heart disease / Myocardial infarction. Optimized protocols for isolation, fixation, and flow cytometric characterization vs. Standard methods (coronary perfusion, density-mediated separation, methanol/acetone fixation) was evaluated on Cellular yield and preservation of antibody binding/cellular morphology. Optimized protocols using low-speed centrifugation, 40-μm filtering, and 1% paraformaldehyde fixation improved immune cell yield and preserved cellular morphology compared to standard methods.
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