We thank the UMass flow cytometry core: Richard Konz, Ted Giehl, Barbara Gosselin, Yuehua Gu and Tammy Krupoch. This work was funded by NIH/NHLBI RO1 HL084168 (M.S.) and NIH CA T32-130807 (J.R.S.). Core resources supported by the Diabetes Endocrinology Research Center grant DK32520 were also used.

1. Harvesting of tissues
<ol>
<li>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. </li>
<li>Cull mice according to appropriate approved protocol (e.g. CO2 inhalation followed by cervical dislocation).</li>
<li>Draw blood by cardiac puncture into EDTA or heparin blood-collection tubes for later analysis, e.g. of hematocrit, reticulocyte count or CBC analysis.</li>
<li>Harvest the spleen an...

<ol>
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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...

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
	<td>Fas-biotin</td>
	<td>BD Pharmingen</td>
	<td>554256</td>
 </tr>
 <tr>
	<td>Streptavidin-APC</td>
	<td>Molecular Probes</td>
	<td>S868</td>
 </tr>
 <tr>
 <td>40 &mu;m sterile cell strainer </td>
 <td>Fisherbrand</td>
 <td>22363547</td>
 </tr>
 <tr>
 <td>Polystyrene round-bottom tubes for FACS staining </td>
 <td>BD Falcon</td>
 <td>352008</td>
 </tr>
 <tr>
 <td>U-bottom 96 well plate </td>
 <td>BD Falcon</td>
 <td>353910</td>
 </tr>
 <tr>
 <td>ChromePure Rabbit IgG</td>
 <td>Jackson ImmunoResearch</td>
 <td>015-000-003</td>
 </tr>
 <tr>
 <td>CD71-FITC (stock 0.5mg/ml)</td>
 <td>BD-Biosciences</td>
 <td>553266</td>
 </tr>
 <tr>
 <td>Ter119-PE (stock 0.2mg/ml) </td>
 <td>BD-Biosciences</td>
 <td>553673</td>
 </tr>
 <tr>
 <td>7AAD </td>
 <td>BD-Biosciences</td>
 <td>559925</td>
 </tr>
 <tr>
 <td>DAPI powder </td>
 <td>Roche</td>
 <td>236276</td>
 </tr>
 <tr>
 <td>FITC Rat Anti-Mouse CD41 MWReg30</td>
 <td>BD Pharmingen </td>
 <td>553848</td>
 </tr>
 <tr>
 <td>FITC Rat Anti-Mouse CD45R/B220 RA3-6B2</td>
 <td>BD Pharmingen </td>
 <td>553087</td>
 </tr>
 <tr>
 <td>FITC Rat Anti-Mouse CD411b/Mac-1 M1/70</td>
 <td>BD Pharmingen </td>
 <td>557396</td>
 </tr>
 <tr>
 <td>FITC Rat Anti-Mouse Ly-6G and Ly-6C (Gr-1) RB6-8C5</td>
 <td>BD Pharmingen </td>
 <td>553126</td>
 </tr>
 <tr>
 <td>FITC Hamster Anti-Mouse CD3e 145-2C11 </td>
 <td>BD Pharmingen </td>
 <td>553061</td>
 </tr>
 <tr>
 <td>APC BrdU Flow kit</td>
 <td>BD Pharmingen</td>
 <td>557892</td>
 </tr>
 <tr>
 <td>Annexin V-biotin</td>
 <td>BD Pharmingen</td>
 <td>556418</td>
 </tr>
 </tbody>
</table>

identification and analysis of mouse erythroid progenitors using the cd71/ter119 flow-cytometric assay

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.

Watch this Scientific Journal Video about Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay at JoVE.com

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

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

identification-analysis-mouse-erythroid-progenitors-using-cd71ter119

Research

JoVE Journal

Biology

31.7K visualizações.  University of Massachusetts Medical School. Um método de citometria de fluxo para a identificação e análise molecular da diferenciação estágio específico progenitores eritróides murino e precursores, diretamente na medula recém-colhidas do mouse óssea, baço ou o fígado fetal. O ensaio baseia-se na superfície celular marcadores CD71, Ter119 e tamanho da célula.