Name: Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry (Video) | JoVE Journal
Uploaded: 2014-06-06T13:00:00.000+00:00
Duration: 9 min 40 s
Description: Learn multi-spectral imaging flow cytometry for identifying enucleating erythroblast subpopulations in mouse bone marrow and spleen cells. This method enables detailed visualization of erythropoiesis.

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5 minutes

The authors thank the Research Flow Cytometry Core at Cincinnati Children&#8217;s Hospital Research Foundation and Richard Demarco, Sherree Friend, and Scott Mordecai from the Amnis Corporation (part of EMD Milllipore) for expert technical support. This work was supported by the National Institutes of Health grants K08HL088126 and R01HL116352 (T.A.K.) and P30 DK090971 (Y.Z.).

1. Long-term In vitro Erythropoiesis Culture (Ex vivo Erythroid Differentiation Culture Protocol by Giarratana et al.8, Modified and Adapted for Mouse Cells)
This is a 3-step long-term in vitro erythropoiesis protocol. In the first step (days 0-4) 2 x 105 cells/ml are placed in erythroblast growth medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (Epo). In the second step (days 5-6), cells are resuspended at 2 x 105 cells/ml and co-cultured on adherent stroma cells (MS5) in fresh erythroblast growth medium supplemented only with Epo. In the third step (days 7-9), cells are cultured on a layer of MS-5 cells in fresh erythroblast growth medium without cytokines up to enucleation (Figure 1A).
All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Cincinnati Children&#8217;s Hospital Medical Center.
<ol>
	<li>Harvest of bones and isolation of low-density bone marrow cells
	<ol>
		<li>Add 2 ml sterile IMDM containing 2% fetal bovine serum (FBS) in a 15-ml conical tube and keep on ice.</li>
		<li>Euthanize a 2-6 month old wild type C57/BL6 mouse (along with or without genetically-targeted mouse of interest) following institution-approved protocol (e.g. CO2 inhalation, followed by cervical dislocation).</li>
		<li>Isolate pelvic bones, femurs, and tibiae of both legs using forceps and scalpel, add them to the tube containing IMDM+2% FBS and keep on ice.</li>
		<li>Add 1 ml IMDM+2% FBS in a sterile flow-cytometry tube and flush bones using forceps and a tuberculin syringe with a 25-G&#160;x 5/8&#34; needle. Flush IMDM+2% FBS through the bones a few times gently (by aspirating ~500 &#181;l from the cell suspension and flushing it again through the bone), and collect the bone marrow cells into the flow-cytometry tube. Flushing is complete when bones appear white.</li>
		<li>Filter cell-suspension through a 40-&#956;m cell strainer set on top of a 50-ml conical tube. Wash cell strainer with IMDM+2% FBS and using the same medium adjust final volume of cell suspension to 5&#160;ml.</li>
		<li>Prepare low-density bone marrow cells (LDBM) by density gradient centrifugation: carefully layer the 5&#160;ml of&#160;cell-suspension on 5 ml of density gradient cell separation medium 1.083 g/ml in a 15-ml tube and spin at 750 x g for&#160;25 min at room temperature (RT) with no brake/low acceleration.</li>
		<li>Transfer supernatant (everything up to about the 2-ml mark of the 15-ml tube in order to acquire buffy coat) to a new 15-ml tube and perform one more wash with IMDM+2% FBS at 525 x g for&#160;5 min at&#160;RT.</li>
		<li>Aspirate supernatant and lyse any remaining red blood cells (RBC) by suspending the pellet in 3 ml RBC lysis buffer for 5 min at&#160;RT. If a greater purity of erythroblasts is required at the final step (e.g. for biochemical studies), purify LDBM cells further at this step to Linneg cells by magnetic separation.</li>
	</ol>
	</li>
	<li>Ex vivo erythroid differentiation culture protocol starting from LDBM or Linneg cells (Figure 1A)
	<ol>
		<li>Add 7 ml IMDM+2% FBS, spin at 525 x g for&#160;5 min at&#160;RT and resuspend the pelleted cells in 2 ml erythroblast growth medium (EGM), consisting of: 
		a. StemPro-34 medium, containing 
		b. 2.6% StemPro-34 medium supplement, 
		c. 20% BIT-9500, 
		d. 900 ng/ml ferrous sulfate, 
		e. 90 ng/ml ferric nitrate, 
		f. 100 units/ml penicillin/streptomycin, 2 mM L-glutamine 
		g. 10-6 M hydrocortisone 
		h. and freshly added cytokines: 
		i) 100 ng/ml SCF, 
		ii) 5 ng/ml IL-3, and 
		iii) 3 IU/ml Epo.</li>
		<li>Count the cells using an automated cell counter or manually at a microscope using a hemocytometer.
		</li>
		<li>Plate in a 6-well cell-culture plate at a concentration of 5 x 105 cells/well to a final volume of 2.5 ml in EGM (2 x 105 LDBM cells/ml) and incubate at 37 &#176;C (this is considered as day #0 of culture).
		</li>
		<li>Change medium by aspirating 1.5 ml of the supernatant, spinning at 525 x g for&#160;5 min at&#160;RT, resuspending in 1.5 ml of fresh medium containing all 3 cytokines and adding back to the wells every day on days #2, 3, 4, to optimize proliferative phase. On day 3, remove all cells, count and split into appropriate number of wells in order to sustain well cell concentrations of ~2 x 105 cells/ml. Maintain culture at 37 &#176;C/5% CO2.</li>
		<li>On day 4, plate MS5 cells (murine stromal cell line) to wells of a new cell-culture 6-well plate. The MS5 cell-culture medium is &#945;-MEM containing 20% FBS, 100 units/ml penicillin/streptomycin, and 2 mM L-glutamine.</li>
		<li>On day 5, count number of cells (now significantly enriched in erythroblasts) in each well of the original culture plate using an automated cell counter or manually at a microscope using a hemocytometer.</li>
		<li>Lift all cells from each well and transfer to 15-ml conical tubes, spin down at 525 x g for&#160;5 min at&#160;RT, aspirate supernatant and dissolve pellets with fresh EGM containing only Epo (3 IU/ml) to a concentration of 2 x 105 cells/ml.</li>
		<li>Aspirate supernatant from the wells where MS-5 cells were plated the previous day (targeted to 70-80% confluency at the time of co-culture) and add the erythroid cells in these wells in EGM containing only Epo (although not their medium of choice, MS-5 cells survive well in EGM for several days).</li>
		<li>Change medium adding fresh EPO on day #6.</li>
		<li>Change medium on day #7, using EGM with no added cytokines. On days 7 through 9, test samples for enucleation with flow cytometry after staining with anti-Ter119 and Syto-16 daily and proceed to staining samples for Multi-Spectral Imaging Flow Cytometry (as detailed in section 3). 
		Note: Before plating and on day 2, 4, 6, and 7 of culture, monitor cultured cells for erythroblast enrichment and differentiation with flow cytometry by assessing surface markers CD44 and Ter119 vs size (FSC).9 Alternatively CD71, Ter119, and FSC can also be used10,11.</li>
	</ol>
	</li>
</ol>
2. Fast Enucleation Assay, According to the Protocol Described by Yoshida et al.12 with Modifications (Figure 1B)
<ol>
	<li>Stress erythropoiesis induction and stroma cell preparation for in vitro erythropoiesis culture
	<ol>
		<li>Anesthetize a 2-6 month old wild type C57/BL6 mouse per IACUC guidelines (e.g. with isoflurane solution). Ensure that the mouse has been adequately anesthetized by checking for absence of reflexive responses to a gentle hind-paw pinch and for regular respirations during the procedure. Use vet ophthalmic ointment on eyes to prevent dryness while under anesthesia.</li>
		<li>Induce stress erythropoiesis via tail bleeding to a final volume of 500 &#956;l. Using an insulin syringe, inject equal volume of normal saline intraperitoneally to assure fluid resuscitation of the animal (hold animal in Trendelenburg position before injecting and inject in the lower abdomen so as not to damage internal organs).</li>
		<li>Do not leave the mouse unattended until it has regained motor control as indicated by the animal starting to move around the cage and being able to stand and walk without falling. The phlebotomized mouse is placed in a cage without other mice.</li>
		<li>After 2 days, plate MS-5 cells in wells of a 24-well cell-culture plate. Incubate MS-5 cells at 37 &#176;C/5% CO2 in MS5 cell medium (a-MEM containing 20% FBS, 100 units/ml penicillin/streptomycin, and 2 mM L-glutamine) with the goal to be 70-80% confluent in plate wells after 48 hr.</li>
	</ol>
	</li>
	<li>Harvest of spleen and processing of splenocytes
	<ol>
		<li>Four days (96 hr) after stress erythropoiesis induction, euthanize previously bled mouse through IACUC-approved protocol (e.g. CO2 inhalation followed by cervical dislocation).</li>
		<li>Harvest spleen and put it in a 15-ml conical tube containing IMDM 2% FBS and keep on ice.</li>
		<li>Back in the laboratory, in the tissue culture hood under sterile conditions, invert spleen-containing tube on a 40-&#956;m cell strainer set on top of a 50-ml tube. Crush spleen using the plunger of a 5-ml plastic syringe.</li>
		<li>Wash cell strainer with IMDM+2% FBS and using the same medium, adjust the final volume of cell suspension to 5 ml.</li>
		<li>Carefully layer the 5 ml splenocyte suspension on 5 ml density gradient cell separation medium 1.083 g/ml in a 15-ml tube and spin at 750 x g for&#160;25 min at&#160;RT with no brake/low acceleration.</li>
		<li>Transfer supernatant (solution down to about the 2-ml mark of the 15-ml tube in order to acquire buffy coat) to a new 15-ml tube and perform one more wash with IMDM+2% FBS at 525 x g for&#160;5 min at&#160;RT.</li>
		<li>Aspirate supernatant and lyse red blood cells by suspending the pellet in 3 ml RBC lysis buffer for 5 min at&#160;RT.</li>
		<li>Add 7 ml IMDM+2% FBS to wash cells and to dilute and neutralize the RBC lysis buffer and spin at 525 x g for&#160;5 min at&#160;4&#160;&#176;C.</li>
		<li>Aspirate supernatant and suspend pelleted cells in 2 ml of erythroblast growth medium (EGM).</li>
	</ol>
	</li>
	<li>Culture of the isolated low-density splenocytes, enriched in erythroblasts, on plastic (first stage of fast in vitro erythropoiesis culture)
	<ol>
		<li>Count cells on an automated cell counter or manually by a hemocytometer. Usual number of cells isolated per spleen at this stage is ~15 x 106.</li>
		<li>Suspend cells further in EGM containing the cytokines (at final concentrations): SCF 50 ng/ml, IL-3 5 ng/ml, and Epo 2 U/ml.</li>
		<li>Plate 1-5 x 106 cells in a final volume of 1 ml/well (same number of cells per well depending on total number of cells) of a 24-well cell-culture plate and incubate O/N at 37&#160;&#176;C/5% CO2.</li>
	</ol>
	</li>
	<li>Culture of erythroblasts on MS5 cells (second stage of fast in vitro erythropoiesis culture
	<ol>
		<li>Aspirate supernatant from the plastic well and lift the cells (highly enriched in erythroblasts) by adding 2 ml of&#160;cold 10 mM EDTA in PBS for 5 min, on ice to each well.</li>
		<li>Put cells in fresh tubes, wash once in EGM (without cytokines) and resuspend in the same.</li>
		<li>Plate 5 x 105-1 x 106 cells in a 1-2 ml volume to each MS5 cell-coated well of a 24-well plate. If pharmacological inhibitors are being used in the experiment, add them at appropriate concentrations now.</li>
		<li>Incubate for 6 to 8 hr&#160;(time guided by microscopic observation of approximately 30%-40% enucleation in the untreated WT sample). The binding of erythroblasts to MS-5 cells accelerates their enucleation.</li>
		<li>Lift cells from each well by adding 2 ml of cold PBS + 10 mM EDTA, for 5 min, on ice. Along with the erythroblasts, MS-5 cells will also be collected but these can later be easily excluded during flow cytometric analysis as FSChi Ter119- cells.</li>
		<li>At this stage, cells can be fixed and stained for analysis by Multi-Spectral Imaging Flow Cytometry.</li>
	</ol>
	</li>
</ol>
3. Staining of Erythroblasts for Localization of Intracellular Proteins or Lipid Rafts During Enucleation by Multi-spectral Imaging Flow Cytometry&#160;
<ol>
	<li>Wash cells in PBS, spin 525 x g for&#160;5 min at&#160;RT and aspirate supernatant.</li>
	<li>Fix cells by resuspending cell pellets in 500 &#956;l of 3.7% formaldehyde in PBS for 15 min (fixation time may vary depending on the antigen being probed) and pipetting gently.</li>
	<li>Transfer to 1.5-ml plastic centrifuge tubes and incubate for 15 min at&#160;RT.</li>
	<li>Spin on a bench microcentrifuge 2,000 x g for&#160;20 sec, aspirate supernatant and perform one wash by adding 500 &#956;l PBS to each tube and pipetting gently.</li>
	<li>Spin on a bench microcentrifuge 2,000 x g for&#160;20 sec, aspirate supernatant and keep tubes on ice for at least 15 min. Permeablization steps 3.6-3.9 should be done quickly and efficiently, and following spinning/aspiration at RT, cell pellets should immediately be put back on ice, in order for cells to better retain their integrity.</li>
	<li>Take acetone solutions out from -20 &#176;C freezer and put on ice. Permeabilize cells by resuspending cell pellets first in 500 &#956;l ice-cold 50% acetone (1:1 with dH2O) and pipetting gently.</li>
	<li>Spin on bench microcentrifuge 2,000 x g for&#160;20 sec, aspirate supernatant and resuspend cell pellets in 500 &#956;l&#160;ice-cold&#160;100% acetone by pipetting gently.</li>
	<li>Spin on bench microcentrifuge 2,000 x g for&#160;20 sec, aspirate supernatant and resuspend cell pellets once more in 500 &#956;l&#160;ice-cold&#160;50% acetone by pipetting gently.</li>
	<li>Spin on bench microcentrifuge 2,000 x g for&#160;20 sec, aspirate supernatant and wash cells once in cold FACS buffer (PBS + 0.5% BSA) by pipetting gently.</li>
	<li>Prepare labeling cocktail with antibodies or markers for the molecules of interest: 0.1 U/100 &#956;l AF488-phalloidin for F-actin staining and 1 &#956;l/100 &#956;l Ter119-PECy7 for erythroid cell staining. Alternative or additional staining can also be done using AF-488-anti-&#946;-tubulin antibody (1:50), AF-594&#8211;conjugated cholera toxin subunit B to label lipid rafts (1:200), anti-pMRLC (Ser19) primary antibody for the phosphorylated myosin regulatory light chain (1:50), followed by anti-rabbit AF-488&#8211;conjugated secondary antibody (1:400), and anti-&#947;-tubulin primary rabbit antibody (1:100) followed by anti-rabbit AF-555-conjugated secondary antibody (1:300).</li>
	<li>Following supernatant aspiration, resuspend cell pellets in 100 &#956;l of the marker cocktail, pipette gently and incubate for 30 min at&#160;RT.</li>
	<li>Prepare FACS buffer containing 2.5 &#956;M of the nuclear stain Draq5.</li>
	<li>Wash cells in FACS buffer, spin down on bench microcentrifuge 2,000 x g for&#160;20 sec, aspirate supernatant and resuspend in 60 &#956;l FACS buffer containing Draq5.</li>
	<li>Run samples on the imaging flow cytometer to collect at least 10,000 events per experiment, compensate the raw data files as previously published13,&#160;and analyze results as shown in Figure 2, using the analysis software specific to the imaging flow cytometer.</li>
</ol>

<ol><li>McGrath, K. E., Kingsley, P. D., Koniski, A. D., Porter, R. L., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enucleation+of+primitive+erythroid+cells+generates+a+transient+population+of+%26%2334%3Bpyrenocytes%26%2334%3B+in+the+mammalian+fetus%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Enucleation of primitive erythroid cells generates a transient population of &#34;pyrenocytes&#34; in the mammalian fetus.</a> Blood. 111, 2409-2417 (2008).</li>
<li>Chasis, J. A., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erythroblastic+islands%3A+niches+for+erythropoiesis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Erythroblastic islands: niches for erythropoiesis.</a> Blood. 112, 470-478 (2008).</li>
<li>Koury, S. T., Koury, M. J., Bondurant, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytoskeletal+distribution+and+function+during+the+maturation+and+enucleation+of+mammalian+erythroblasts%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts.</a> J Cell Biol. 109, 3005-3013 (1989).</li>
<li>Ji, P., Jayapal, S. R., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enucleation+of+cultured+mouse+fetal+erythroblasts+requires+Rac+GTPases+and+mDia2%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2.</a> Nat Cell Biol. 10, 314-321 (2008).</li>
<li>Keerthivasan, G., Small, S., Liu, H., Wickrema, A., Crispino, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicle+trafficking+plays+a+novel+role+in+erythroblast+enucleation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Vesicle trafficking plays a novel role in erythroblast enucleation.</a> Blood. 116, 3331-3340 (2010).</li>
<li>McGrath, K. E., Bushnell, T. P., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multispectral+imaging+of+hematopoietic+cells%3A+where+flow+meets+morphology%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Multispectral imaging of hematopoietic cells: where flow meets morphology.</a> J Immunol Methods. 336, 91-97 (2008).</li>
<li>Konstantinidis, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signaling+and+cytoskeletal+requirements+in+erythroblast+enucleation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Signaling and cytoskeletal requirements in erythroblast enucleation.</a> Blood. 119, 6118-6127 (2012).</li>
<li>Giarratana, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+generation+of+fully+mature+human+red+blood+cells+from+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.</a> Nat Biotechnol. 23, 69-74 (2005).</li>
<li>Chen, K., Liu, J., Heck, S., Chasis, J. A., An, X., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resolving+the+distinct+stages+in+erythroid+differentiation+based+on+dynamic+changes+in+membrane+protein+expression+during+erythropoiesis%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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. , (2009).</li>
<li>Kalfa, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rac1+and+Rac2+GTPases+are+necessary+for+early+erythropoietic+expansion+in+the+bone+marrow+but+not+in+the+spleen%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen.</a> Haematologica. 95, 27-35 (2010).</li>
<li>Koulnis, M., Pop, R., Porpiglia, E., Shearstone, J. R., Hidalgo, D., Socolovsky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+analysis+of+mouse+erythroid+progenitors+using+the+CD71%2FTER119+flow-cytometric+assay%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay.</a> J Vis Exp. , (2011).</li>
<li>Yoshida, H., Kawane, K., Koike, M., Mori, Y., Uchiyama, Y., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphatidylserine-dependent+engulfment+by+macrophages+of+nuclei+from+erythroid+precursor+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells.</a> Nature. 437, 754-758 (2005).</li>
<li>Ortyn, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitivity+measurement+and+compensation+in+spectral+imaging%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Sensitivity measurement and compensation in spectral imaging.</a> Cytometry A. 69, 852-862 (2006).</li>
</ol>

The authors declare no competing financial interests.

In recent years the study of erythroblast enucleation has gained increasing momentum&#160;since it is the step in in vitro erythropoiesis cultures that is most difficult to reproduce efficiently in order to achieve successful, large-scale production of red blood cells ex vivo.&#160;Up until recently, the study of erythroblast enucleation utilized mainly fluorescence microscopy and flow cytometry methods.&#160;Fluorescence microscopy methods, albeit helpful in identifying participating molecules, require...

Terminal differentiation within the erythroid lineage in mammals concludes with the dramatic process of enucleation, through which the orthochromatic erythroblast expels its membrane-encased nucleus (pyrenocyte)1, generating a reticulocyte2. The exact mechanism of this process, which is also the rate-limiting step of successful, large-scale, production of red blood cells in vitro, is not yet fully elucidated. The localization of key proteins and structures within enucleating erythroblasts relies on the use of fluorescent and electron microscopy3-5. This tedious process typically results in the identification of a limited number of enucleation events and does not always allow meaningful statistical analysis. Expanding on a method of erythroblast identification described previously by McGrath et al.6, we have developed a novel approach of identifying and studying enucleation events by Multi-Spectral Imaging Flow Cytometry (multiparameter high-speed cell imaging in flow, a method that combines fluorescent microscopy with flow cytometry)7, which can provide a sufficient number of observations to obtain measurements and perform statistical analysis.
Here, we describe first two in vitro erythropoiesis culture methods used in order to synchronize erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.
Samples are run on an imaging flow cytometer and the collected cells are gated appropriately to identify orthochromatic erythroblasts6. Orthochromatic erythroblasts are then analyzed based on their aspect ratio, as measured in brightfield imaging, versus their value for the parameter delta centroid XY Ter119-DNA, which is defined as the distance between the centers of the areas stained for Ter119 and DNA, respectively. The population of cells with low aspect ratio and high delta centroid XY Ter119/DNA is highly enriched in enucleating cells.&#160;Using wild-type (WT) erythroblasts versus erythroblasts with Mx-Cre mediated conditional deletion of Rac1 on Rac2-/- or combined Rac2-/-; Rac3-/- genetic background and this novel analysis protocol of multi-spectral imaging flow cytometry, we recently demonstrated that enucleation resembles asymmetric cytokinesis and that the formation of an actomyosin ring regulated in part by Rac GTPases is important for enucleation progression7.

<table><tbody><tr><td>&amp;alpha;MEM medium</td><td>CellGro</td><td>15-012-CV</td><td></td></tr><tr><td>IMDM medium</td><td>Hyclone (Thermo Scientific)</td><td>SH30228.01</td><td></td></tr><tr><td>Stempro-34 SFM</td><td>GIBCO (Life Tech)</td><td>10640</td><td></td></tr><tr><td>Stempro-34 nutrient supplement</td><td>GIBCO (Life Tech)</td><td>10641-025</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>512450</td><td></td></tr><tr><td>BIT9500</td><td>Stemcell Technologies</td><td>09500</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP-1600-100</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Hyclone (Thermo Scientific)</td><td>SH30028.02</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Hyclone (Thermo Scientific)</td><td>SV30010</td><td></td></tr><tr><td>L-glutamine</td><td>Hyclone (Thermo Scientific)</td><td>SH30590.01</td><td></td></tr><tr><td>Isothesia (Isoflurane)</td><td>Butler-Schein</td><td>029405</td><td></td></tr><tr><td>Histopaque 1.083 mg/ml</td><td>Sigma</td><td>10831</td><td></td></tr><tr><td>BD Pharmlyse (RBC lysis buffer)</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>534064</td><td></td></tr><tr><td>Formaldehyde</td><td>Fisher Scientific</td><td>BP 531-500</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H4001</td><td></td></tr><tr><td>Stem Cell Factor (SCF)</td><td>Peprotech</td><td>250-03</td><td></td></tr><tr><td>Interleukin-3 (IL-3)</td><td>Peprotech</td><td>213-13</td><td></td></tr><tr><td>EPOGEN Epoetin Alfa (Erythropoietin, EPO)</td><td>AMGEN</td><td>available by pharmacy</td><td></td></tr><tr><td>CD44-FITC antibody</td><td>BD Pharmingen</td><td>553133</td><td></td></tr><tr><td>CD71-FITC antibody</td><td>BD Pharmingen</td><td>553266</td><td></td></tr><tr><td>Ter119-PECy7 antibody</td><td>BD Pharmingen</td><td>557853</td><td></td></tr><tr><td>Phalloidin-AF488</td><td>Invitrogen (Life Technologies)</td><td>A12379</td><td></td></tr><tr><td>&amp;beta;-tubulin-AF488 antibody</td><td>Cell Signaling</td><td>#3623</td><td></td></tr><tr><td>anti-rabbit AF488-secondary antibody</td><td>Invitrogen (Life Technologies)</td><td>A11008</td><td></td></tr><tr><td>anti-rabbit AF555-secondary antibody</td><td>Invitrogen (Life Technologies)</td><td>A21428</td><td></td></tr><tr><td>AF594-cholera toxin B subunit</td><td>Invitrogen (Life Technologies)</td><td>C34777</td><td></td></tr><tr><td>pMRLC (Ser19) antibody</td><td>Cell Signaling</td><td>#3671</td><td></td></tr><tr><td>&amp;gamma;-tubulin antibody</td><td>Sigma</td><td>T-3559</td><td></td></tr><tr><td>Syto16</td><td>Invitrogen (Life Technologies)</td><td>S7578</td><td></td></tr><tr><td>Draq5</td><td>Biostatus</td><td>DR50200</td><td></td></tr><tr><td>Ferrous sulfate</td><td>Sigma</td><td>F7002</td><td></td></tr><tr><td>Ferric nitrate</td><td>Sigma</td><td>F3002</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP120500</td><td></td></tr><tr><td>15-ml tubes</td><td>BD Falcon</td><td>352099</td><td></td></tr><tr><td>50-ml tubes</td><td>BD Falcon</td><td>352098</td><td></td></tr><tr><td>6-well plates</td><td>BD Falcon</td><td>353046</td><td></td></tr><tr><td>24-well plates</td><td>BD Falcon</td><td>351147</td><td></td></tr><tr><td>Flow tubes</td><td>BD Falcon</td><td>352008</td><td></td></tr><tr><td>Tuberculin syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Insulin syringe</td><td>BD</td><td>329461</td><td></td></tr><tr><td>Syringe needle 25-G 5/8</td><td>BD</td><td>305122</td><td></td></tr><tr><td>Capped flow tubes</td><td>BD</td><td>352058</td><td></td></tr><tr><td>40-&amp;mu;m cell strainer</td><td>BD Falcon</td><td>352340</td><td></td></tr><tr><td>Scalpel (disposable)</td><td>Feather</td><td>2975#21</td><td></td></tr><tr><td>FACS Canto Flow Cytometer</td><td>BD</td><td></td><td></td></tr><tr><td>ImagestreamX Mark II Imaging Flow Cytometer</td><td>AMNIS (EMD Millipore)</td><td></td><td></td></tr><tr><td>Image Data Exploration and Analysis Software (IDEAS) version 4.0 and up.</td><td>AMNIS (EMD Millipore)</td><td></td><td></td></tr><tr><td>Hemavet 950 Cell Counter</td><td>Drew Scientific</td><td>CDC-9950-002</td><td></td></tr><tr><td>NAPCO series 8000WJ Incubator</td><td>Thermo scientific</td><td></td><td></td></tr><tr><td>Allegra X-15R Centrifuge</td><td>Beckman Coulter</td><td>392932</td><td></td></tr><tr><td>Mini Mouse Bench centrifuge</td><td>Denville</td><td>C0801</td><td></td></tr></tbody></table>

identification of a murine erythroblast subpopulation  enriched in enucleating events by multi-spectral imaging flow cytometry

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

First, cells are analyzed based on their Brightfield Aspect Ratio (the ratio of the length of their minor versus their major axis) and their Brightfield Area (indicative of their size). Events with a Brightfield Area value lower than 20 and higher than 200 are mostly debris and cell aggregates, respectively, and are excluded from the analysis (Figure 2A). Single cells (gate “R1”) are then analyzed based on their value for the Gradient RMS parameter, which indicates sharpness of image. Gate “R2” is create...

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

The overall goal of this procedure is to facilitate the identification of enucleation events with an aurn Erythroblast subpopulation. The first step is to create a population enriched in nucleating events. This is accomplished by in vitro erythropoiesis culture, either long-term starting from low density bone marrow cells isolated from the femurs, tibia, and pelvic bones of a non-stressed mouse or short-term culture.Starting with ple cytes from the spleen of a mouse subjected previously to phlebotomy in order to mount stress erythropoiesis. The cultured cells at the final step of the culture are fixed, permeated, and stained with the fluorescent markers of interest. These cells are run on an imaging flow cytometer and then analyzed to identify the cells undergoing enucleation in the population of nucleating cells signaling and cytoskeletal components that regulate and mediate the process of enucleation can be identified.The main advantage of this technique over existing methods such as fluorescent microscopy, is that the significantly larger number of nucleating cells can be observed and analyzed in a short amount of time. This method can help to answer key questions in the erythropoiesis field, such as what molecular mechanisms are involved in erythroblast nucleation. To differentiate erythroid cells from low density bone marrow cells begin by adding one milliliter of culture media to a sterile flow cytometry tube.Then attach a 25 gauge by five eighths inch needle to a tuberculin syringe and gently flush fresh culture media through the harvested femurs and tibia a few times into the flow cytometry tube. After adjusting cell suspension volume to five milliliters with IMDM, carefully layer onto five milliliters of 1.083 grams per milliliter density gradient cell separation medium in a 15 milliliter tube. Transfer everything down to the two milliliter mark into a new 15 milliliter tube and wash the collected buffy coat with fresh culture media lice the red blood cells in three milliliters of red blood cell lysis buffer for five minutes at room temperature after washing cells in culture media and resus suspending the pellet in the same plate, the cells in a six well cell culture plate at a concentration of five times 10th of the fifth cells per well to a final volume of 2.5 milliliters in growth media and incubate the cells at 37 degrees Celsius five days after plating the culture.Count the number of erythroblasts in each well of the original culture plate and then transfer each well into corresponding individual 15 milliliter conical tubes. After spinning down the cells, aspirate the supernatants and resuspend the pellets in fresh growth media containing only erythropoietin to a concentration of two times 10 of the fifth cells per milliliter. Then aspirate the supernatant from a plate of MS five cells and add the erythroid cells to the wells.Two days later, replace the supernatant with fresh growth media without cytokines toul erythroblasts from low density PLE cytes. Use the plunger from a five milliliter syringe to macerate the spleen through a 40 micron strainer into a 50 milliliter tube. Wash the cell strainer with growth media and adjust the final volume of cell suspension to five milliliters.Then after separating the cell layers by density gradient as just demonstrated, lice the red blood cells and wash the cytes in seven milliliters of culture media. Then Resus suspend the pellet in growth media supplemented with cytokines and incubate the cells at one to five times 10 to the six cells in a final volume of one milliliter per well of a 24 well cell culture plate overnight at 37 degrees Celsius and 5%carbon dioxide. The following day.Transfer cells to wells containing adhered MS five cells as previously described and analyze after six to eight hours, collect the erythroblasts from the culture wells and fix them in 500 microliters of 3.7%formaldehyde in PBS Resus, suspending the pellet with gentle pipetting. After 15 minutes, spin the cells at 2000 GS for 20 seconds in a micro centrifuge, aspirate the supernatant and re suspend the pellet in 500 microliters of PBS. After spinning down the cells in the micro centrifuge, again, aspirate the supernatant again and place the cells on ice for at least 15 minutes.Put minus 20 degrees Celsius, stored acetone solutions on ice. Then quickly resuspend and spin down the cells in a series of acetone washes as indicated after the fax buffer wash, incubate the cell pellets in 100 microliters of the appropriate antibody cocktail for 30 minutes at room temperature. Then after washing the cells in fax buffer again, re suspend the sustained pellets in 60 microliters of fax buffer containing drac five and run the samples on an imaging flow cytometer collecting at least 10, 000 events per experimental sample.Then start the analysis by selecting cells based on their brightfield aspect ratio, the ratio of the length of their minor versus their major axis and their brightfield area, which is indicative of their size. Events with a brightfield area value lower than 20 and higher than 200 are mostly debris and cell aggregates respectively and are excluded from the analysis. The then analyze the single cells based on their value for the gradient RMS parameter, which indicates the sharpness of image create a second gate containing the cells with the gradient RMS value greater than the 50th percentile in order to select the images taken well in focus.Next gate, the cells based on their size as measured by their brightfield area and their positivity for the erythroid marker TER one 19 as measured by the TER one 19 fluorescent stain mean pixel parameter cells very low or very high for TER one 19 are either non erythroid or remaining cell aggregates respectively and are excluded from the analysis in the next step. The cells are selected based on their DR five aspect ratio intensity, the ratio of the minor versus the major axis of their nucleus and the intensity of their d dr five expression. DR five negative cells mostly enucleated cells such as reticulocytes and red blood cells and cells with a low drag five aspect ratio.Mostly doublets are excluded from the analysis gait. The drag five positive DNA positive cells, which are mostly erythroblasts at this point. These erythroblasts are then analyzed based on their TER one 19 area, which indicates the size of the cell and their TER one 19 mean pixel area or density of TER one 19 expression, which indicates the brightness of their TER one 19 staining.Ortho chromatic erythroblasts are recognized as small TER one 19 high cells. Finally, a subpopulation of the ortho chromatic erythroblasts is characterized by their low brightfield aspect ratio and their high delta sentu XY TUR one 19. DR five as defined by the distance between the center of the incipient TUR one 19 positive reticulocyte and the center of the DR five positive nucleus along with antibody against her one 19 and the DNA stain DRAAK five.The cells are typically also stained to identify the localization of f actin during erythroblast and nucleation. Of note, the progression of enucleation can be visualized as cells with a decreasing aspect ratio. That is cells that become increasingly elongated and with an increasing delta OID xy TUR one 19 drag five are observed.F actin concentrates at the cleavage furrow during in nucleation and then dissipates once the nucleus is extruded. Other proteins and structures of interest can also be stained with the appropriate antibodies or fluorescent markers to allow imaging of their roles during enucleation. For example, polarized microtubule formation can be visualized in wild type ortho chromatic erythroblasts prior to a nucleation, but not in erythroblasts treated with colchicine.Inhibition of tubulin polymerization by colchicine diminishes the cell polarization as demonstrated by measuring the parameter delta OID XYB FDR five between the center of the cell body seen in the Brightfield channel and the center of the nuclear staining achieved with DR five. Following this procedure, other methods such as fluorescence microscopy can be performed to answer questions concerning the interactions between different cell types involved in erythropoiesis such as those observed in Erythroblast islands. After watching this video, you should have a good understanding of how to visualize enucleation events in an erythroblast enriched population.

identification-murine-erythroblast-subpopulation-enriched-enucleating

Research

JoVE Journal

Biology

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Learn multi-spectral imaging flow cytometry for identifying enucleating erythroblast subpopulations in mouse bone marrow and spleen cells. This method enables detailed visualization of erythropoiesis.

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

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