A method to increase reproducibility in adult ventricular myocyte sizing and flow cytometry: Avoiding cell size bias in single cell preparations
Abstract
Flow cytometry (FCM) of ventricular myocytes (VMs) is an emerging technology in adult cardiac research that is challenged by the wide variety of VM shapes and sizes. Cellular variability and cytometer flow cell size can affect cytometer performance. These two factors of variance limit assay validity and reproducibility across laboratories. Washing and filtering of ventricular cells in suspension are routinely done to prevent cell clumping and minimize data variability without the appropriate standardization. We hypothesize that washing and filtering arbitrarily biases towards sampling smaller VMs than what actually exist in the adult heart. To determine the impact of washing and filtering on adult ventricular cells for cell sizing and FCM. Left ventricular cardiac cells in single-cell suspension were harvested from New Zealand White rabbits and fixed prior to analysis. Each ventricular sample was aliquoted before washing or filtering through a 40, 70, 100 or 200μm mesh. The outcomes of the study are VM volume by Coulter Multisizer and light-scatter signatures by FCM. Data are presented as mean±SD. Myocyte volumes without washing or filtering (NF) served as the "gold standard" within the sample and ranged from 11,017 to 46,926μm...Continue Reading