Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

Summary

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly –harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

Abstract

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation.

The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

Protocol

1. Harvesting of tissues

1. Prepare tubes containing 2 to 5 ml cold staining buffer (phosphate-buffered saline (PBS) with added 0.2% BSA and 5mM glucose). Keep tubes on ice prior to tissue harvest.
2. Cull mice according to appropriate approved protocol (e.g. CO₂ inhalation followed by cervical dislocation).
3. Draw blood by cardiac puncture into EDTA or heparin blood-collection tubes for later analysis, e.g. of hematocrit, reticulocyte count or CBC analysis.
4. Harvest the spleen and bones, placing tissues from each mouse in a separate tube, prepared in step 1. Easy access to spleen is from the left side. For the bone-marrow, har....

Discussion

The flow-cytometric methodology allows simultaneous investigation of any cellular function that may be detected with a fluorescence-conjugated specific antibody or ligand, including cell surface markers, protein expression, cell survival, cell signaling using phospho-specific antibodies ³ and cell cycle status. These measurements may be made in each of a number of differentiation-stage specific subsets, in the context of freshly isolated erythropoietic tissue. This method therefore allows assessment of functio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fas-biotin	BD Biosciences	554256
Streptavidin-APC	Molecular Probes, Life Technologies	S868
40 μm sterile cell strainer	Fisher Scientific	22363547
Polystyrene round-bottom tubes for FACS staining	BD Biosciences	352008
U-bottom 96 well plate	BD Biosciences	353910
ChromePure Rabbit IgG	Jackson ImmunoResearch	015-000-003
CD71-FITC (stock 0.5mg/ml)	BD Biosciences	553266
Ter119-PE (stock 0.2mg/ml)	BD Biosciences	553673
7AAD	BD Biosciences	559925
DAPI powder	Roche Group	236276
FITC Rat Anti-Mouse CD41 MWReg30	BD Biosciences	553848
FITC Rat Anti-Mouse CD45R/B220 RA3-6B2	BD Biosciences	553087
FITC Rat Anti-Mouse CD411b/Mac-1 M1/70	BD Biosciences	557396
FITC Rat Anti-Mouse Ly-6G and Ly-6C (Gr-1) RB6-8C5	BD Biosciences	553126
FITC Hamster Anti-Mouse CD3e 145-2C11	BD Biosciences	553061
APC BrdU Flow kit	BD Biosciences	557892
Annexin V-biotin	BD Biosciences	556418