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 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, harvest one or both femurs. Keep harvested tissue on ice. </li>
<li>If desired, weigh the spleen. Mice undergoing an erythropoietic stress response are likely to show a significant increase in spleen weight. </li>
</ol>
2. Preparation of spleen cells
<ol>
<li>Using a pre-moistened 3 ml syringe plunger, gently push the spleen, or part of the spleen (ideally, 0.1 gram or less, equivalent to approximately 108 cells) through a 40 &mu;m sterile cell strainer (Fisherbrand catalog number 22363547 or other) placed on top of a 50 ml conical tube. Keep the tube on ice during this procedure. Wash the cells through the strainer with a total of 2 ml staining buffer. </li>
<li>Gently pipette the strained cell suspension to break up any small clumps. If necessary, re- strain the cells.</li>
<li>Wash the cells twice by centrifugation and re-suspend in cold buffer.</li>
<li>Count the cells using a hemocytometer. Typical yields are 1-2 x 108 cells/spleen. For flow-cytometry analysis, aliquote 1 to 2 million cells per sample, either into FACS staining tubes (BD Falcon polystyrene round-bottom tubes, 352008) or U-bottom 96 well plate (BD Falcon 353910). Sample staining volume is 200 &mu;l, to a final cell concentration 0.5 to 1 x 107 cells/ ml or 1-2 x 106 cells/ 200 &mu;l sample.</li>
</ol>
3. Preparation of bone-marrow cells
<ol>
<li>Prepare 1 or 3 ml syringes with an attached 26G needle, pre-filled with cold staining buffer.</li>
<li>Remove muscles attached to the femur so as to visualize the bone clearly. </li>
<li>Using sharp surgical scissors, snip off both ends of the femur, as close as possible to the ends of the bone. This should reveal a small hole at each cut end, leading into the bone marrow cavity, which runs through the length of the femur.</li>
<li>Using the pre-filled syringes in step 1, insert the needle through one of these holes, and gently flush the marrow out through the hole at the other end, into a tube.</li>
<li>Dissociate the flushed cells by gentle pipetting, and strain through a 40 &mu;m strainer as for the spleen above (see section 2.1).</li>
<li>Wash cells twice by centrifugation in cold staining buffer</li>
<li>Count the cells and resuspend as in section 2.4. Typical yields are approximately 107 cells per femur.</li>
</ol>
4. Preparation of fetal liver cells
<ol>
<li>To prepare timed-pregnant female mice, set up mice for mating in the evening; examine for vaginal plugs before 10 am the following day; the day on which the vaginal plug is detected is considered day 0.5. Veterinary staff may be able to assist investigators who are unfamiliar with this technique to identify pregnant females. Pregnancy can also be determined by monitoring mouse weight.</li>
<li>Timed &#x2013;pregnant female mice are culled on days 12.5 to 14.5 of pregnancy. The uterine horns are removed into a Petri-dish containing ice-cold culture medium or staining buffer.</li>
<li>Embryos are removed from each uterus and the fetal liver is dissected. A dissecting microscope is required for embryonic day 12.5 (E12.5) or younger. </li>
<li>Livers may be dissociated mechanically by pipetting in buffer, and are processed either individually in 96 well plates, or pooled together, depending on experimental requirements. </li>
<li>A fetal liver at E13.5 has ~107 cells. Cells are washed twice in staining buffer and resuspended at 1-2 x 106 cells/ 200 &mu;l sample for flow cytometric analysis. </li>
</ol>
5. Antibody staining for flow cytometry
<ol>
<li>Prepare a primary antibody staining pre-mix to be used for all samples, except for control samples, containing the following:
<ul>
<li>ChromePure Rabbit IgG (Jackson, 015-000-003), to a final concentration of 200&mu;g/ml. Check the stock concentration on the bottle (it can vary). This is used to block Fc receptors in mouse cells; alternative species that may be used for this are mouse IgG or rat IgG. Species choice is determined by the potential presence in the staining protocol of secondary antibodies directed against primary rat/mouse/rabbit antibodies, in which case those species cannot be used as blocking antibodies. Alternatively, 5% rabbit serum may also be used in place of purified IgG. A further alternative is the use of monoclonal antibodies or Fab fragments directed at the mouse Fc receptors. The basic protocol below does not include any secondary antibodies and so IgG of any of the three species may be used.</li>
<li>CD71-FITC, diluted 1:200 (stock 0.5mg/ml, BD-Biosciences, 553266)</li>
<li>Ter119-PE, diluted 1:200 (stock 0.2mg/ml, BD-Biosciences, 553673)</li>
<li>Any additional antibodies directed at surface epitopes of interest, e.g. antibodies directed at Fas or FasL (see 1,2). Mix the antibody solution gently by inverting the tube 2-3 times. </li>
</ul>
</li>
<li>Add 200 &mu;l of the pre-mix to each cell sample and gently re-suspend the cells.</li>
<li>Prepare control cell samples as follows:
<ul>
<li>'Unstained': these cells are left in staining buffer and provide the background autofluorescence of the cells.</li>
<li>'Single color' controls: one such control is required for each antibody/color used in the protocol. The cells in these controls are stained either with a directly conjugated primary antibody, or with both a primary antibody and a conjugated secondary antibody. These controls are used to correct for spectral overlap between channels.</li>
<li>'Fluorescence minus one' (FMO) controls: one such control is required for each antibody/color in the protocol: cells are stained with all the colors/antibodies in the protocol except for the color/antibody for which this is the FMO control. The FMO control for a particular channel provides the true background for that channel. It may include non-specific antibody of the same isotype, and conjugated with the same fluorescence mark as the test antibody (isotype control). </li>
</ul>
</li>
<li>Incubate samples and relevant controls in the primary antibody stain on ice for 45' to 1 hour in the dark (put aluminium foil cover on ice-bucket). </li>
<li>At the end of incubation, wash the cells by adding 3ml of staining buffer to each sample tube and spin for 3' to 5' at approximately 400 x g at 4&deg;C. If using 96 well plates, wash the cells three times in a volume of 200 &mu;l.</li>
<li>If relevant, apply secondary antibody stain. Apply and wash as for the first antibody stain.</li>
<li>If relevant, a stain with Annexin V is applied at the end of incubation, using a hepes buffer as in the manufacturer's instructions. This stain is applied for 15 minutes at room-temperature, or for 1 hour on ice.</li>
<li>Cells are re-suspended for flow-cytometry analysis in staining solution containing a cell-impermeable DNA dye, to exclude dead cells. Several DNA dyes are available, including Propidium Iodide, 7-amino-actinomycin D (7AAD), or DAPI. The choice from amongst these depends on the available channels, given the channels taken up for specific antibody staining, and channels available on the flow-cytometer. 7AAD is obtained from BD-Biosciences (559925) and used according to the manufacturer's instructions. For DAPI staining, make a stock of 1mg/ml in dimethylformamide (DMF) from powder (Roche, Cat # 236276), keep at -20&deg;C, and dilute 1:10,000 to 1:15,000 in staining buffer. </li>
</ol>
6. Flow-cytometric sorting
<ol>
<li>Cells are labeled with antibodies against CD71, Ter119, lineage markers, and a viability dye as described for flow-cytometric analysis (section 5). Cell concentration during labeling may be increased to 5 x 107/ml. </li>
<li>Mix an equal volume of each of the following antibodies to make the lineage master mix:
<ul>
<li>FITC Rat Anti-Mouse CD41 MWReg30, BD Pharmingen 553848</li>
<li>FITC Rat Anti-Mouse CD45R/B220 RA3-6B2, BD Pharmingen 553087</li>
<li>FITC Hamster Anti-Mouse CD3e 145-2C11, BD Pharmingen 553061</li>
<li>FITC Rat Anti-Mouse CD11b/Mac-1 M1/70, BD Pharmingen 557396</li>
<li>FITC Rat Anti-Mouse Ly-6G and Ly-6C (Gr-1) RB6-8C5, BD Pharmingen 553126 </li>
<li>Use the master mix at 1:80 (This is equivalent to 1:400 dilution of each individual antibody stocks, which are all 0.5 mg/ml).</li>
</ul>
</li>
<li>Use low-pressure sorting conditions and wide nozzles. For the Aria (BD Biosciences) we use 100 &mu; nozzle, 20 psi pressure.</li>
<li>Collection buffer: PBS with added 20% Fetal Bovine Serum. </li>
<li>To check the purities of the sorted populations, re-run a small aliquot from each sample in a buffer that contains a viability dye (7AAD or similar).</li>
</ol>
7. Representative Results: 
CD71/Ter119 staining of adult bone-marrow or spleen identifies a developmental sequence of four subsets, labeled ProE, EryA, EryB and EryC (Figure 1) 1. Morphologically, these correspond to increasingly mature erythroblasts. Figure 1 illustrates the gating sequence at the data analysis stage, which discards very small event (including nuclei, red cells), aggregated cells and dead cells. 
Expression of cell-surface proteins may be measured simultaneously for each of these subsets, by adding the relevant antibodies at the same time as Ter119 and CD71 staining. Figure 1 shows an example of cell-surface expression of the death receptor Fas 1. This measurement was carried out in mice injected with Epo, or in control mice injected with saline. It is apparent that Epo suppresses Fas expression in the EryA population in vivo 1. 
Expression of intracellular proteins or cell cycle status may also be measured for cells in each subset. Figure 2 illustrates representative cell cycle analysis of freshly harvested bone marrow cells. These measurements require, in addition to cell surface staining with CD71 and Ter119, the fixation and permeabilization of cells for intracellular labeling (see Discussion section). 
In fetal liver, non-erythroid cells are first excluded by gating on 'Lin-' cells that are negative for CD41, Mac-1, Gr-1, B220 and CD3 (Figure 3). The remaining cells are sub-divided into 6 subsets, S0 to S5. The precise pattern of cells in fetal liver is dependent on embryonic age (see Discussion section). A representative cell cycle analysis of the S3 subset in E13.5 fetal liver is shown (Figure 4). 
<img src="/files/ftp_upload/2809/2809fig1.jpg" alt="Figure 1"/>
 Figure 1. The CD71/Ter119 erythroid subsets in mouse spleen. A. Gating strategy: Spleen cells were processed and labeled with antibodies directed at CD71, Ter119 and Fas. This figure shows the analysis strategy following the data acquisition step. Histogram I shows all acquired events. The diagonal gate represents events that are likely to be single cells, excluding doublets or larger aggregates. Cells in this gate are further analyzed in histogram II. Here very small events, likely nuclei or debris, are excluded. The gated cells are shown in histogram III, where DAPI-positive cells, that are likely membrane-permeable apoptotic cells, are excluded from further analysis. Histogram IV shows the resulting population of viable spleen cells. The ProE gate contains CD71highTer119intermediate cells. Ter119high cells are further analyzed in histogram V. Here CD71high cells are subdivided into less mature, large 'EryA' erythroblasts (CD71highTer119highFSChigh) and smaller, more mature 'EryB' erythroblasts (CD71highTer119highFSClow). The most mature erythroblast subset is EryC (CD71lowTer119highFSClow). Histogram VI shows cell-surface Fas expression, specifically in the EryA subset, in mice in the basal state (injected with saline), and mice injected with a single dose of Epo. Staining with Fas antibody was carried out simultaneously with the CD71/Ter119 staining. B. Cytospin preparations of cells sorted from each of the indicated subsets. Cells were stained with Giemsa and with Diaminobenzidine, the latter generates a brown stain with hemoglobin. Cytospin data was originally published in Liu et al., Blood. 2006 Jul 1;108(1):123-33. Epub 2006 Mar 9.
<img src="/files/ftp_upload/2809/2809fig2.jpg" alt="Figure 2"/> 
Figure 2. Cell cycle analysis of CD71highTer119high erythroblasts in mouse bone marrow. Mice were injected intraperitoneally with BrdU, and spleen or bone-marrow were harvested 30 to 60 minutes later. Cells were fixed and permeabilized and in addition to being stained for CD71 and Ter119, were stained for BrdU incorporation into their replicating DNA with a monoclonal antibody directed at BrdU (fixation, permeabilization and BrdU-staining protocol was according to manufacturer's instruction). BrdU-positive cells are in S-phase of the cycle. Interphase cells are BrdU-negative and may be resolved into G1 or G2/M phases, using the DNA dye 7AAD.
<img src="/files/ftp_upload/2809/2809fig3.jpg" alt="Figure 3"/> 
Figure 3. CD71/Ter119 erythroid subsets in mouse fetal liver. A. Gating strategy: Fetal liver cells were labeled for CD71, Ter119, and a cocktail of FITC &#x2013;labeled antibodies directed at non-erythroid lineage markers ('Lin'). Viable cells (7AAD-negative) were analyzed for Lin expression, and the Lin- cells are further subdivided into the S0 to S5 erythroid subsets. Younger, E13 fetal liver is composed of less mature erythroblasts, shown by the absence of cells in the mature S4/S5 subsets. B. Cytospin preparations of cells sorted from each of the indicated subsets. Cells were stained with Giemsa and with Diaminobenzidine, the latter generates a brown stain with hemoglobin. Cytospin data was originally published in Pop et al., PLoS Biol 8(9): e1000484. doi:10.1371/journal.pbio.1000484 .
<img src="/files/ftp_upload/2809/2809fig4.jpg" alt="Figure 4"/> 
Figure 4. Cell cycle analysis of fetal liver erythroid subsets. Pregnant mice were injected with BrdU, and fetal livers were harvested 30 to 60 minutes later, fixed, permeabilized, and stained with antibodies against CD71, Ter119 and BrdU. Cell cycle status of S3 cells is shown.

<ol>
	<li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+Fas-FasL+coexpression+by+erythropoietin+mediates+erythroblast+expansion+during+the+erythropoietic+stress+response+in.">Suppression of Fas-FasL coexpression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in.</a> 108, 123-133 (2006).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+Autoregulation+by+FAS+Mediates+Robust+Fetal+Erythropoiesis.">Negative Autoregulation by FAS Mediates Robust Fetal Erythropoiesis.</a> PLoS Biol. 5, e252-e252 (2007).</li><li>Krutzik, P. O., Hale, M. B., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+murine+immunological+signaling+network+with+phosphospecific+flow+cytometry.">Characterization of the murine immunological signaling network with phosphospecific flow cytometry.</a> J Immunol. 175, 2366-2373 (2005).</li><li>Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ineffective+erythropoiesis+in+Stat5a(-/-)5b(-/-)+mice+due+to+decreased+survival+of+early+erythroblasts.">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98, 3261-3273 (2001).</li><li>Guihard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+ERK1+is+a+negative+regulator+of+the+adult+steady-state+splenic+erythropoiesis.">The MAPK ERK1 is a negative regulator of the adult steady-state splenic erythropoiesis.</a> Blood. 115, 3686-3694 (2010).</li><li>Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+erythroid+chaperone+that+facilitates+folding+of+alpha-globin+subunits+for+hemoglobin+synthesis.">An erythroid chaperone that facilitates folding of alpha-globin subunits for hemoglobin synthesis.</a> J Clin Invest. 117, 1856-1865 (2007).</li><li>Chen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythroid+dysplasia,+megaloblastic+anemia,+and+impaired+lymphopoiesis+arising+from+mitochondrial+dysfunction.">Erythroid dysplasia, megaloblastic anemia, and impaired lymphopoiesis arising from mitochondrial dysfunction.</a> Blood. 114, 4045-4053 (2009).</li><li>Chen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+distinct+stages+in+erythroid+differentiation+based+on+dynamic+changes+in+membrane+protein+expression+during+erythropoiesis.">Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.</a> Proc Natl Acad Sci U S A. 106, 17413-17418 (2009).</li><li>McGrath, K. E., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+hematopoietic+cells:+where+flow+meets+morphology.">Multispectral imaging of hematopoietic cells: where flow meets morphology.</a> J Immunol Methods. 336, 91-97 (2008).</li><li>Pop, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+key+commitment+step+in+erythropoiesis+is+synchronized+with+the+cell+cycle+clock+through+mutual+inhibition+between+PU.1+and+S-phase+progression.">A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression.</a> PLoS Biol. 8, (2010).</li><li>Borsook, H., Lingrel, J. B., Scaro, J. L., Millette, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+haemoglobin+in+relation+to+the+maturation+of+erythroid+cells.">Synthesis of haemoglobin in relation to the maturation of erythroid cells.</a> Nature. 196, 347-350 (1962).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by University of Massachusetts Medical School IACUC committee.

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 ...

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32.1K Views.  University of Massachusetts Medical School. 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.

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...