Name: Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells (Video) | JoVE Journal
Uploaded: 2025-07-11T14:50:00.000+00:00
Duration: 5 min 10 s
Description: Learn a cost-effective method to purify and expand mouse bone marrow mesenchymal stem cells using flow cytometry-based phenotyping. This protocol yields homogeneous BMSCs with strong growth and differentiation potential for preclinical research.

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5 minutes

This work was supported by grants from the National Key R&#38;D Program of China (2020YFE0201600) and the National Natural Science Foundation of China (82174408 and 82374477).

<ol><li>Chu, D. -T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+update+on+the+progress+of+isolation%2C+culture%2C+storage%2C+and+clinical+application+of+human+bone+marrow+mesenchymal+stem%2Fstromal+cells%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">An update on the progress of isolation, culture, storage, and clinical application of human bone marrow mesenchymal stem/stromal cells.</a> Int J Mol Sci. 21 (3), 708(2020).</li>
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<li>Maridas, D. E., Rendina-Ruedy, E., Le, P. T., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+culture%2C+and+differentiation+of+bone+marrow+stromal+cells+and+osteoclast+progenitors+from+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice.</a> J Vis Exp. (131), e56750(2018).</li>
<li>Chu, D. -T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adipose+tissue+stem+cells+for+therapy%3A+An+update+on+the+progress+of+isolation%2C+culture%2C+storage%2C+and+clinical+application%5BTitle%5D)+AND+%22J+Clin+Med%22%5BJournal%5D)">Adipose tissue stem cells for therapy: An update on the progress of isolation, culture, storage, and clinical application.</a> J Clin Med. 8 (7), 917(2019).</li>
<li>Qin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+in+fibrotic+diseases%E2%80%94the+two+sides+of+the+same+coin%5BTitle%5D)+AND+%22Acta+Pharmacol+Sin%22%5BJournal%5D)">Mesenchymal stem cells in fibrotic diseases—the two sides of the same coin.</a> Acta Pharmacol Sin. 44 (2), 268-287 (2023).</li>
<li>Carbone, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+cervical+dislocation+as+a+humane+euthanasia+method+in+mice%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Assessing cervical dislocation as a humane euthanasia method in mice.</a> J Am Assoc Lab Anim Sci. 51 (3), 352-356 (2012).</li>
<li>Swamydas, M., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation%2C+purification+and+labeling+of+mouse+bone+marrow+neutrophils+for+functional+studies+and+adoptive+transfer+experiments%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments.</a> J Vis Exp. (77), e50586(2013).</li>
<li>Grossner, T., Haberkorn, U., Hofmann, J., Gotterbarm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+different+basal+cell+culture+media+upon+the+osteogenic+response+of+hMSCs+evaluated+by+99mTc-HDP+labeling%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Effects of different basal cell culture media upon the osteogenic response of hMSCs evaluated by 99mTc-HDP labeling.</a> Int J Mol Sci. 23 (11), 6288(2022).</li>
<li>Janda, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surrogate+Wnt+agonists+that+phenocopy+canonical+Wnt+and+%CE%B2-catenin+signalling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling.</a> Nature. 545 (7653), 234-237 (2017).</li>
<li>Bernar, A., Gebetsberger, J. V., Bauer, M., Streif, W., Schirmer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+Alizarin+Red+S+assay+by+enhancing+mineralization+of+osteoblasts%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Optimization of the Alizarin Red S assay by enhancing mineralization of osteoblasts.</a> Int J Mol Sci. 24 (1), 723(2022).</li>
<li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Restoration+of+osteogenic+differentiation+of+bone+marrow+mesenchymal+stem+cells+in+mice+inhibited+by+cyclophosphamide+with+psoralen%5BTitle%5D)+AND+%22Chin+J+Tissue+Eng+Res%22%5BJournal%5D)">Restoration of osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide with psoralen.</a> Chin J Tissue Eng Res. 29 (1), 16-23 (2025).</li>
<li>Papaccio, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concise+review%3A+Cancer+cells%2C+cancer+stem+cells%2C+and+mesenchymal+stem+cells%3A+Influence+in+cancer+development%5BTitle%5D)+AND+%22Stem+Cells+Transl+Med%22%5BJournal%5D)">Concise review: Cancer cells, cancer stem cells, and mesenchymal stem cells: Influence in cancer development.</a> Stem Cells Transl Med. 6 (12), 2115-2125 (2017).</li>
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<li>Soleimani, M., Nadri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.</a> Nat Protoc. 4 (1), 102-106 (2009).</li>
<li>Abdallah, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+and+reliable+protocol+for+long-term+culture+of+murine+bone+marrow+stromal+%28mesenchymal%29+stem+cells+that+retained+their+in+vitro+and+in+vivo+stemness+in+long-term+culture%5BTitle%5D)+AND+%22Biol+Proced+Online%22%5BJournal%5D)">A simple and reliable protocol for long-term culture of murine bone marrow stromal (mesenchymal) stem cells that retained their in vitro and in vivo stemness in long-term culture.</a> Biol Proced Online. 21 (1), 3(2019).</li>
<li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+cell+sequencing+reveals+gene+expression+signatures+associated+with+bone+marrow+stromal+cell+subpopulations+and+time+in+culture%5BTitle%5D)+AND+%22J+Transl+Med%22%5BJournal%5D)">Single cell sequencing reveals gene expression signatures associated with bone marrow stromal cell subpopulations and time in culture.</a> J Transl Med. 17 (1), 23(2019).</li>
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The authors declare no conflicts of interest in relation to this study.

Stem cell therapy has garnered considerable interest due to its potential to revolutionize regenerative medicine. Preclinical studies have provided substantial evidence supporting the clinical application of stem cells, particularly BMSCs13. However, challenges such as low cell yield, contamination, and slow growth have impeded their clinical application. Therefore, there is an urgent need for research focusing on the standardization of BMSC preparation processes to enhance cell yield and function...

Bone marrow mesenchymal stem cells (BMSCs) are known for their multipotent characteristics and are widely used in the treatment of various diseases due to their self-renewable, differentiation, and immunomodulatory properties1,2. BMSCs are readily obtainable, capable of extensive expansion, exhibit immunosuppressive properties, and do not trigger immediate immune reactions3, making them appealing for tissue engineering applications and effective carriers for cell therapy.
In the bone marrow, two main stem cell populations and their progenies, the hematopoietic stem cells and the BMSCs, are the primary residents4. Traditionally, BMSCs are obtained through targeted bone marrow aspiration and identified as plastic-adherent cells in vitro. However, this method is insufficient to differentiate BMSCs from other bone marrow subpopulations, including endothelial cells, pericytes, leukocytes, and hematopoietic stem cells5. Therefore, an easy and effective protocol for isolation of BMSCs is needed.
The current work details the separation and purification of BMSCs through their physical adherence to plastic cell culture plates. Flow cytometry experiments were conducted on third passage BMSCs using the surface markers CD29, CD44, and Sca-1, as outlined by the International Society for Cell &#38; Gene Therapy (ISCT) Mesenchymal Stromal Cell (MSC) committee6. According to ISCT criteria, mouse MSCs are characterized by positivity for CD29, CD44, and stem cell antigen 1 (Sca-1), and negativity for hematopoietic cell marker CD45 and endothelial cell marker CD31. Additionally, BMSCs should be multipotent, demonstrating trilineage differentiation into osteoblasts, chondrocytes, and adipocytes. In this experiment, osteogenesis was induced in the extracted BMSCs, with ALP staining performed on day 7 and an alizarin red S assay on day 21 to assess differentiation. Adipogenic differentiation of isolated and purified BMSCs was also induced, with Oil Red O staining performed on day 21 to assess adipogenesis.

<table><tbody><tr><td>1mL syringe(22G syringe needle)</td><td>KINDLY GROUP(KDL)</td><td>60017031</td><td></td></tr><tr><td>1-Step NBT/BCIP&amp;nbsp;</td><td>Pierce Biotechnology, Inc.</td><td>34042</td><td>Alkaline phosphatase staining</td></tr><tr><td>10% neutral buffered formalin</td><td>Wexis</td><td>5200250</td><td>cell fixation</td></tr><tr><td>6-well plate</td><td>Labselect</td><td>11110</td><td>Cell Culture</td></tr><tr><td>24-well plate</td><td>Labselect</td><td>11310</td><td>Cell Culture</td></tr><tr><td>40 &amp;mu;m Cell Strainer</td><td>Cornig</td><td>352340</td><td>Filtration to remove agglomerates</td></tr><tr><td>50 mL syringe</td><td>KINDLY GROUP(KDL)</td><td>60017040</td><td></td></tr><tr><td>75% ethanol&amp;nbsp;</td><td>Shanghai NianYue Biotechnology Co., Ltd</td><td>31009001</td><td>Disinfect the skin of mice</td></tr><tr><td>Alizarin Red S</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A5533</td><td>Calcium Deposits and Calcified Nodules Detection</td></tr><tr><td>APC CD44 Monoclonal Antibody (clone:IM7)</td><td>eBioscience</td><td>17-0441-82</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>APC Rat IgG2b kappa Isotype Control (Clone:eB149/10H5)</td><td>eBioscience</td><td>17-4031-82</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>Autoclave</td><td>Japan Hiryama Company</td><td>HVE-50</td><td>Sterilization surgical instruments</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Technology (China)Co., Ltd.</td><td>300583057</td><td>Cell Culture</td></tr><tr><td>Cytoflex flowmetry</td><td>Beckman Coulter&amp;nbsp;</td><td>A00-1-1102</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>dexamethasone</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D4902</td><td>BMSCs osteogenic differentiation</td></tr><tr><td>Dish (100mm)</td><td>Corning</td><td>430167</td><td>Cell Culture</td></tr><tr><td>Dish (60mm)</td><td>Corning</td><td>430166</td><td>Sample placment during transplantation&amp;nbsp;</td></tr><tr><td>Disinfectant cotton balls</td><td>Shanghai Honglong Industrial Co., Ltd.</td><td>20230627</td><td>Disinfect the skin of mice</td></tr><tr><td>Disposable sterile gloves</td><td>Shanghai Honglong Industrial Co.,Ltd</td><td>YT21131</td><td>Sterile operation</td></tr><tr><td>Double lion Irradiated Rodent Diet</td><td>Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd.</td><td>GB 14924.3</td><td>Animal feed</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Shanghai Biosun Sci&amp;amp;Tech Co.,Ltd.</td><td>BS-0002-500</td><td>Cell Culture</td></tr><tr><td>FITC Hamster Anti-Rat CD29 (clone:Ha2/5)</td><td>BD Biosciences</td><td>555003</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>FITC Hamster IgM, &amp;lambda;1 Isotype Control (clone:G235-1)</td><td>BD Biosciences</td><td>553960</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>gelatin (0.1%)</td><td>OriCell</td><td>GLT-11301</td><td>enhance the adhesion ability of cells</td></tr><tr><td>IVCs mice cage</td><td>Suzhou Monkey King Animal Experimental Equipment Technology Co., Ltd.</td><td>HH-MMB-2</td><td>Animal barrier</td></tr><tr><td>L-ascorbic acid</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A8960</td><td>BMSCs osteogenic differentiation</td></tr><tr><td>Mark pen</td><td>Zebra Trading (Shenzhen) Co.,Ltd.&amp;nbsp;</td><td>YYST5</td><td>Mark the cellular information on the Petri dish</td></tr><tr><td>MEM Alpha Modification (1X)</td><td>Shanghai NianYue Biotechnology Co., Ltd</td><td>SH30265</td><td>Cell Culture</td></tr><tr><td>Mouse Bone Marrow Mesenchymal Stem Cells Adipogenic Differentiation Kit</td><td>Cyagen</td><td>MUXMX-90031</td><td>BMSCs adipogenic differentiation&amp;nbsp;</td></tr><tr><td>Nikon Eclipse Ti2 Inverted Microscope Synstem</td><td>Japan Nikon Corporatin</td><td>Ti2-U</td><td>Observe the cells and take photos</td></tr><tr><td>Ophthalmic scissors&amp;nbsp;</td><td>Shanghai NianYue Biotechnology Co., Ltd</td><td>Y00030 JZ</td><td>Cut the skin</td></tr><tr><td>Ophthalmic tweezers</td><td>Shanghai NianYue Biotechnology Co., Ltd</td><td>BS-ZER-S-100 Biosharp</td><td>Obtain mouse bone tissue.</td></tr><tr><td>PASTEUR PIPETTE</td><td>Shanghai NianYue Biotechnology Co., Ltd</td><td>BS-XG-03M</td><td>Aspirate the culture medium</td></tr><tr><td>PE Rat Anti-Mouse CD31 (clone:Ha2/5)</td><td>BD Biosciences</td><td>553373</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>PE Rat IgG2a, &amp;kappa; Isotype Control (clone:R35-95)</td><td>BD Biosciences</td><td>553930</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>PE-Cyanine7 Ly-6A/E (Sca-1) Monoclonal Antibody (clone:D7)</td><td>eBioscience</td><td>25-5981-82</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>PE-Cyanine7 Rat IgG2a kappa Isotype Control (clone:eBR2a)</td><td>eBioscience</td><td>25-4321-82</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin</td><td>Shanghai NianYue Biotechnology Co., Ltd</td><td>2585636</td><td>Cell Culture</td></tr><tr><td>PerCP-Cy5.5 Rat Anti-Mouse CD45 (clone:Ha2/5)</td><td>BD Biosciences</td><td>561869</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>PerCP-Cy5.5 Rat IgG2b, &amp;kappa; Isotype Control (clone:A95-1)</td><td>BD Biosciences</td><td>550764</td><td>Flow cytometry&amp;nbsp;</td></tr><tr><td>Scalpels</td><td>Surgical Instrument Factory oShanghai Medical Devices?Group?Co., Ltd.&amp;nbsp;</td><td>J11010-10# JZ</td><td>Isolate the mouse&#039;s back limbs and torso, and remove extraneous tissues from the bone</td></tr><tr><td>Sterile hood</td><td>Thermo Fisher Technology (China)Co., Ltd.</td><td>ECO0.9</td><td>Surgical operation table</td></tr><tr><td>sterile surgical drapes</td><td>Henan Huayu Medical Equipment Co., Ltd.</td><td>20160900</td><td>Provide sterile surgery area</td></tr><tr><td>Tube (50 mL)</td><td>Shanghai Baisai, Biotechnology Co., Ltd.&amp;nbsp;</td><td>BLD-BL2002500</td><td>Store the filtered cell solution</td></tr><tr><td>trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200056</td><td>digest adherent cells</td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G5422</td><td>BMSCs osteogenic differentiation</td></tr></tbody></table>

purification, expansion, and flow cytometry-based phenotyping of mouse derived bone marrow mesenchymal stem cells

Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of stem cells that can self-renew and differentiate into multiple cell lineages. Among them, MSCs found in bone marrow, known as bone marrow mesenchymal stem cells (BMSCs), play a crucial role in osteogenesis, hematopoietic support, homing/migration, and immunosuppression. BMSCs are widely used in cell biology and tissue engineering research due to their potential for cellular therapeutic strategies, as well as their easy availability, genetic stability, and low immunogenicity. Hematopoietic stem cells (HSCs) and BMSCs coexist in bone marrow but have distinct progenitor cells. When extracting cells from bone marrow, multiple cell types are typically obtained, which can lead to heterogeneity in the cell population, potentially causing bias and not meeting high experimental standards. Therefore, it is crucial to develop effective techniques to improve the homogeneity and yield of BMSCs&#160;in vitro. This study presents a detailed protocol for the purification, expansion, and flow cytometry-based phenotyping of BMSCs obtained from BALB/c mice. The homogeneity and stem cell phenotype of third-passage BMSCs were characterized using flow cytometry, demonstrating positivity for CD29, CD44, and Sca-1, negativity for CD45, and CD31, and confirming their osteogenic and adipogenic differentiation ability. This cost-effective protocol provides an easy and efficient method to obtain a sufficient number of homogeneous BMSCs with high proliferation and differentiation potential, which is essential for advancing research in mouse models and extensive preclinical studies.

Mouse-derived BMSCs were isolated and cultured for approximately 2 weeks to generate the third passage (P3) BMSCs (Figure 3). As shown in Figure 3A, cells freshly extracted from the bone marrow remained mononuclear with visible fat droplets. Spindle-shaped cells appeared in the culture dish, reaching a 60%-80% confluency on the 5th culture day (Figure 3B,C). From the P1-P3...

The present protocol describes a cost-effective method to purify, expand, and characterize highly homogeneous bone marrow mesenchymal stem cells (BMSCs) from mice. This approach facilitates the acquisition of large quantities of BMSCs with high proliferation and differentiation potential, supporting mouse models and advancing preclinical research.

Mesenchymal stem cells (MSCs) are a population of stem cells that can self-renew and differentiate into multiple cell lineages. Among them, MSCs found in ...

purification-expansion-flow-cytometry-based-phenotyping-mouse-derived

Research

JoVE Journal

Biology

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Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

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

Isolation of Mouse Megakaryocyte Progenitors

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

Learn a cost-effective method to purify and expand mouse bone marrow mesenchymal stem cells using flow cytometry-based phenotyping. This protocol yields homogeneous BMSCs with strong growth and differentiation potential for preclinical research.

Purification, Expansion, and Flow Cytometry-Based Phenotyping of Mouse Derived Bone Marrow Mesenchymal Stem Cells

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