Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Abstract

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis. We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

EKLF/KLF1-regulated cell cycle exit is essential for erythroblast enucleation

“…Enucleating cells were identified by their asymmetric nuclei (d centroid) within an elongated cell shape. 29 Quantification by median fluorescence intensity analysis of staged image flow data shows that, compared with Eklf 1/1 , Eklf 2/2 cells have no significant differences in surface Kit expression in progenitors, a 50% reduced expression of CD71 at all erythroblast differentiation stages, but only 1% expression of Ter119 in orthochromatic erythroblasts ( Figure 2B-C), demonstrating again that CD71 and Kit but not Ter119 can be used as a suitable surface marker to sort these cells from either source. Together, these imaging flow analyses enable precise quantification of erythroid differentiation status using morphology without relying on the changes in cell surface marker expression.…”

Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation

“…17 In brief, wild-type (WT) mice subjected to phlebotomy (removal of 300 mL blood for 3 consecutive days to induce stress erythropoiesis) were euthanized 3 days after last phlebotomy and the spleens collected. Splenocytes were pelleted at 1600 rpm/3 min in a bench-top centrifuge, fixed in phosphatebuffered saline containing 4% formaldehyde for 15 minutes at room temperature (RT), and permeabilized by consecutive suspensions in icecold 50% acetone, 100% acetone, and again 50% acetone solution.…”

Centrosome function is critical during terminal erythroid differentiation

“…Fluorescence analysis was performed using IDEAS or FlowJo software. Analysis of the enucleating population was performed as described elsewhere (Konstantinidis et al , 2014 ).…”