Experiment Description

ErythronDB is built upon an extensive database of gene expression data. Follow the links below to view the experimental design and explore sample and data relationships.

Investigation Description: View   Download
Sample and Data Relationships: View   Download

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Methods

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Erythroid Cell Fractionation Protocol

The ErythronDB resource provides access to gene expression data obtained from all three lineages of murine erythropoiesis: Primitive, Fetal Definitive, and Adult Definitive. Primitive samples were obtained from a pooling of single-litter yolk sacs or embryonic blood. Fetal Definitive samples were obtained from pooled samples, again from a single litter, of either fetal liver or fetal blood. Adult Definitive samples were not pooled, but obtained from the bone marrow of individual adults.

All samples were then isolated using a FACS protocol to segregated four stages of erythrocyte development: proerythroblast, basophilic erythroblast, polyorthochromatic erythroblast, and reticulocyte. The protocols employed a combination of cell-surface markers and DNA/RNA gates to differentiate among the stages. They are described below.

Primitive

Primitive erythroid cells mature semi-synchronously in the circulation, thus we also used time as a component for their isolation:

Proerythroblast Basophilic Erythroblast Polyorthochromatic Erythroblast Reticulocyte
E9.5 E10.5 E12.5 E15.5

Reticulocytes were isolated form peripheral blood, using scatter characteristics to purify them from co-occurring definitive fetal reticulocytes.

All primitive stages were purified using surface expression of Ter119 as well as utilizing a DNA intercolater (Draq5 or Vybrant Violet, VV) and a stain for RNA (Thiazole Orange, TO).

Definitive

Fetal definitive erythroid cells are produced asynchronously and cells were isolated from E14.5 fetal liver, except for reticulocytes isolated from E15.5 peripheral blood.

Bone marrow-derived adult definitive erythroid cells also are produced asynchronously and were isolated from mature female bone marrow.

Definitive nucleated erythroblasts were staged by surface phenotype, as well as nuclear condensation and RNA content and size.

Proerythroblast Basophilic Erythroblast Polyorthochromatic Erythroblast Reticulocyte
  • Ter119 mid to hight
  • CD117 Positive
  • Large Cells
  • +Ter119
  • -CD117
  • Draq5/VV DNA intercolater
  • +Ter119
  • -CD117
  • TO Stain for RNA
  • +Ter119
  • -DNA
  • +RNA

In practice, the more condensed DNA/lower RNA containing definitive erythroblasts were a mix of both polychromatophilic and orthochromatic erythroblasts, so we collected a single fraction and named it PolyO.

All definitive stages were also filtered for appropriate forward and side scatter characteristics.