Optimized Protocols for Isolation, Fixation and Flow Cytometric Characterization of Leukocytes in Ischemic Hearts.

Optimized Protocols for Isolation, Fixation and Flow Cytometric Characterization of Leukocytes in Ischemic Hearts. Am J Physiol Heart Circ Physiol. 2019 Aug 02;: Authors: Covarrubias R, Ismahil MA, Rokosh G, Hamid T, Accornero F, Singh H, Gumina RJ, Prabhu S, Bansal S Abstract 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 intra-vascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does...
Source: American Journal of Physiology. Heart and Circulatory Physiology - Category: Physiology Authors: Tags: Am J Physiol Heart Circ Physiol Source Type: research