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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32.0K 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 ...

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 ...

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.

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

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, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

Quantitative Analysis of Mitophagy in Murine Pancreatic &amp;#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &amp;#946;-Cells Using Effective and Robust Complementary Approaches 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Quantitative Analysis of Microbial Autoaggregation Using Imaging Flow Cytometry

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Author Spotlight: Advancing Research in Microbial Autoaggregation Using Imaging Flow Cytometry

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

Mesenchymal Stem Cell Sample Preparation, Flow Cytometric Analysis, and Cell Sorting

To begin, label two new fax tubes as mixed tubes one and two. Prepare the cell and antibody suspension as per the given table for flow cytometric analysis. After incubating the tubes for 30 minutes, wash each tube two times with one milliliter of staining buffer.Centrifuge the vortexed tubes at 200 G for 10 minutes at room temperature. Then re-suspend the pellet in 500 microliters of staining buffer. Coat the wells of a 48 well plate with 200 to 500 microliters of FBS, and keep for one hour.Then pipette 200 to 500 microliters of 10%MSC media after removing the residual FBS. To prepare the samples for single cell sorting, pipette one milliliter of FBS into two new fax tubes. Roll the tubes to create an even coat of the FBS on the tube's inner surface.Prepare the cell and antibody suspension in mixed tubes one and two, as per the table, for the sorting experiment. Then incubate the tubes in the dark for 30 minutes at room temperature. After washing with staining buffer, centrifuge the tubes at 200 G for 10 minutes at room temperature.Then re-suspend the pellet in 500 microliters of staining buffer and incubate for 15 minutes in five microliters of DAPI before sorting. To set up the compensation matrix, use the single stain compensation tubes. Click on experiment from the toolbar in the instrument software.Then click on compensation setup, followed by create compensation controls. Next, click on unstained tube, followed by parameters in the cytometer dialogue box. Adjust the voltages by editing the values for each parameter.Click on load in the acquisition dashboard and then click acquire. Drag the P-1 gate to the cell population and apply it across all compensation controls to set the voltage and negative gate for each parameter. Now load the single stain compensation tubes individually.Record the data and save it. Select the current experiment, right click on it, and select calculate compensation values, followed by link and save. Run the mixed tube one and record 10, 000 cells.Set the gates on the population of interest to be sorted with the proper gating strategy. Next, load a collection plate into the instrument and set a target cell number, ranging from 2, 500 to 5, 000 cells per well. Then select the single cell sort purity mask.Collect the sorted cell populations in a collection device, keeping them on ice for the duration of the sorting experiment. Finally, shift the plates into a 5%carbon dioxide incubator to maintain the cultures at 37 degrees Celsius. The multi-parametric flow cytometry assays plots showed no expression of the FITC isotype of CD 90 FITC antibodies.Positive expression of the markers CD-90 and CD-73 and negative expression of CD-45 are comparable with that of their unstained and FMO controls. The immuno phenotypes showed the marker distribution from one of the early passages with the ISCT recommended markers and CD-44, determining the parent populations as MSCs. The late passage sheds were used for single cell sorting of high and dim expressors.The post sorted cells collected in the 48 well plate showed adherence and proliferation, like their parent population. The post sorted cells differentiated in the presence of adipogenic growth factors.

Single-Cell Dissociation of Murine Islets for Flow Cytometric Analysis of &#946;-Cell Mitophagy

Begin by culturing the eyelet samples isolated from regular fat diet or high fat diet mice overnight at 37 degrees Celsius. Design the desired experimental conditions for the study. For each experimental condition, using a pipette, pick 100 eyelets into a six centimeter Petri dish containing two milliliters of eyelet medium.To induce Mitophagy, treat the eyelets in the appropriate Petri dishes with two microliters of 250 nano molar Valinomycin stock for three hours. Isolate the eyelets and using a pipette, transfer the eyelets from each Petri dish to a separate micro centrifuge tube. Then, spin the eyelets at 350 G for one minute at 10 degrees Celsius and using a pipette, discard the supernatant.Wash the obtained sample twice with one milliliter of PBS. Between the washes, spin the sample and discard the supernatant as demonstrated previously. To dissociate eyelets into single cells, add 500 microliters of 0.05%Trypsin prewarmed to 37 degrees celsius, to one sample tube.Pipette the contents of the tube up and down repeatedly until the eyelets are visibly dispersed. Immediately add one milliliter of prewarmed eyelet medium to neutralize trypsin. Spin the samples at 500 G for five minutes at 10 degrees Celsius.Remove the supernatant carefully without disturbing the pellet. Then, wash the samples two times with eyelet flow medium. The cell samples are now ready to be stained for flow cytometric analysis of beta cell mitophagy.