The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.

<ol>
	<li>Forthun, R. B., Hinrichs, C., Dowling, T. H., Bruserud, &. #. 2. 1. 6. ;., Selheim, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+subclassification+of+patients+with+acute+myeloid+leukemia.">The past, present and future subclassification of patients with acute myeloid leukemia.</a> Curr. Pharm. Biotechnol. 17 (1), 6-19 (2016).</li><li>Goldie, H., Butler, C. H., Anderson, M. M., Maxwell, M. C., Hahn, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+characteristics+of+free+C1498+(granulocytic+leukemia)+tumor+cells+in+the+peritoneal+fluid+and+the+blood+of+C57+mice.">Growth characteristics of free C1498 (granulocytic leukemia) tumor cells in the peritoneal fluid and the blood of C57 mice.</a> Cancer Res. 13 (2), 125-129 (1953).</li><li>Tanaka, K. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+an+implanted+murine+myelogenous+leukemia+C1498.">Studies of an implanted murine myelogenous leukemia C1498.</a> Ohio J. Sci. 75 (4), 202-208 (1975).</li><li>Boyer, M. W., Orchard, P. J., Gorden, K. B., Anderson, P. M., Mclvor, R. S., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependency+on+intercellular+adhesion+molecule+recognition+and+local+interleukin-2+provision+in+generation+of+an+in+vivo+CD8++T-cell+immune+response+to+murine+myeloid+leukemia.">Dependency on intercellular adhesion molecule recognition and local interleukin-2 provision in generation of an in vivo CD8+ T-cell immune response to murine myeloid leukemia.</a> Blood. 85 (9), 2498-2506 (1995).</li><li>Labelle, J. L., Truitt, 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=Characterization+of+a+murine+NKT+cell+tumor+previously+described+as+an+acute+myelogenous+leukemia.">Characterization of a murine NKT cell tumor previously described as an acute myelogenous leukemia.</a> Leuk. Lymphoma. 43 (8), 1637-1644 (2002).</li><li>Bradner, W. T., Pindell, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukemia+as+a+screen+for+cancer+chemotherapeutic+agents.">Myeloid leukemia as a screen for cancer chemotherapeutic agents.</a> Cancer Res. 26 (4), 375-390 (1966).</li><li>Lin, J. 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M., Fountebouni, A., Sullivan, R., Zuckerman, K., Ryan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+normal+murine+hematopoiesis+by+leukemic+cells.">Inhibition of normal murine hematopoiesis by leukemic cells.</a> N.Engl.J. Med. 299 (2), 71-75 (1978).</li><li>Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Subsets of myeloid-derived suppressor cells in tumor-bearing mice.</a> J. Immunol. 181 (8), 5791-5802 (2008).</li><li>Loder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+development+in+the+spleen+takes+place+in+discrete+steps+and+is+determined+by+the+quality+of+B+cell+receptor-derived+signals.">B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.</a> J. Exp. Med. 190 (1), 75-89 (1999).</li><li>Robbins, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extramedullary+hematopoiesis+generates+Ly-6C(high)+monocytes+that+infiltrate+atherosclerotic+lesions.">Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions.</a> Circulation. 125 (2), 364-374 (2012).</li></ol>

The authors declare that they have no competing financial interests.

In previous studies, the C1498 cell line was described as an inducer of acute granulocytic5, myelomonocytic6 or NKT7 cell leukemia. However, demonstrative data in the literature were either absent or incomplete. The protocol presented here uses different techniques, such as flow cytometry, immunofluorescence, MGG staining and cytochemical assays, to characterize cultured C1498 cells and to determine the nature of the leukemia that is induced in mice after they are injected.
When we phenotyped in vitro cultured C1498 cells after immunostaining and flow cytometry analyses were performed, we observed some limitations because these cells expressed few cell surface hematopoietic markers that have been previously described in the literature6,7. In agreement with our results, Labelle et al. did not observe the cell surface expression of mature TCR on C1498 cells using flow cytometry staining. However, they considered them to be a NKT cell line after they detected CD3&#949; and TCRV&#946;8.2 mRNAs7. We also observed the intracellular expression of TCRV&#946; chains and CD3&#949; molecules in most of the cells (&#62; 70%), but their hematopoietic lineages could not be determined because there was also concomitant intracellular expression of the Mac-3 molecule.
Myeloperoxidase, MGG staining and assessments to analyze functional esterases using cytochemistry demonstrated that the C1498 cell line had a myeloid origin and was composed of monoblasts and myeloblasts. These results were concordant with the percentage of Mac-3+ cells that were obtained in flow cytometry staining analysis. Although not quantitative, these steps represent key experiments to be performed. Indeed, they remain, so far, the best existing methods for characterizing the lineage and differentiation stage of hematopoietic cells that express no or few specific phenotypic markers.
Flow cytometry staining was helpful for demonstrating the development of acute leukemia in congenic mice after C1498 cells were intravenously injected. The CD45.2+ C1498 cells that infiltrated into the peripheral blood and various organs were isolated, and their frequencies were determined. Quantification was also performed to analyze inherent medullary and splenic cells after immunophenotyping. Limitations were encountered when the C1498 cell phenotype was examined in organs as they expressed few hematopoietic markers (only a few of them were B220+). To define the nature of the observed acute leukemia, May-Gr&#252;nwald Giemsa staining and an analysis of the activities of monocytic and granulocytic esterases were performed using bone marrow. The results showed that C1498 cells preserved their myeloblastic and monoblastic morphology and function, revealing the onset of myelomonocytic leukemia.
In consideration for the critical steps described in this protocol, particular attention should be given to pH when performing cytochemical reactions and MGG staining because errors in pH can lead to incorrect interpretations of results. For instance, &#945;-naphthyl butyrate esterase activity is specific to monocytic cells only at a pH of 6.0 because granulocytes and lymphocytes can also stain positive for this test at higher pH values. Fixating the cells is not recommended before performing MGG staining, and we showed that only CAF fixation provided satisfactory results when performing esterases cytochemical reactions using C1498 cells. To preserve the expression of the CD115 molecule and its detection by flow cytometry, all of the samples (e.g., blood, bone marrow, and spleen cells) should be kept on ice during the procedure. If no staining is observed in flow cytometry or/and immunofluorescence experiments, the reference of the antibodies, their storage recommendations and their dilutions should be checked. The references specified in the materials/equipment table have been selected for flow cytometry or immunofluorescence applications. The primary/secondary antibodies or their conjugated fluorophores might have lost their activity due to inappropriate storage (e.g., exposure to light or heat), improper dilution, extensive freezing/thawing or the use of contaminated buffers. Run positive controls to ensure that they are working properly. Use mouse bone marrow or spleen-derived cells that are known to express the proteins of interest. To avoid high background and non-specific staining, make sure that the cells are washed properly and kept at high humidity (for immunofluorescence) and that the antibodies are diluted as instructed. Use the same concentration and dilution for the isotype control antibody and the primary antibody to accurately determine the background level in the sample. For esterase cytochemistry experiments, the reagents can be tested by using positive and negative control slides containing purified mouse splenic granulocytic (Ly6G+) and monocytic (CD115+) cells.
The procedure described in this study showed that many of the leukemic features observed in mice after the injection of C1498 cells shared common hallmarks with human acute myelomonocytic leukemia11,12. The invaded leukemic cells resulted in a reduction of mature and immature (progenitors and precursors) medullary hematopoietic cells. C1498 cells are present at high frequency (&#62; 20%) in the peripheral blood, as are monocytic cells. Hepatomegaly and splenomegaly were observed to result from the infiltration of leukemic cells, and significant increases in B lymphocytes and myeloid cells were also observed to accompany splenomegaly. Thrombopenia was also observed when blood platelets numbers were estimated using a hematology analyzer.
It was shown, using in vitro experiments, that C1498 cells inhibit normal murine hematopoiesis by secreting soluble factors13. In several tumor mouse models, immature myeloid cells (including monocytic and granulocytic cells) have also been shown to migrate from the bone marrow to the spleen, where they inhibit anti-tumor specific T cell activation and proliferation14. Thus, the reduction in hematopoietic cells that was observed in the bone marrow could have resulted from either a deficiency in hematopoiesis and/or from their emigration. This latter mechanism could explain the presence of monocytosis in the peripheral blood or the observation of enlarged myeloid fractions in the spleen. It is also conceivable that these cells could have been derived from improved splenic hematopoiesis. Indeed, under steady-state conditions, some subsets of splenic B cells were identified as precursors of mature B lymphocytes15. Moreover, under inflammatory conditions, medullary stem and progenitors cells have been shown to relocate to the spleen to induce the production of mature monocytes16. This protocol does not allow us to draw conclusions regarding the mechanisms that are involved in the development of leukemia, and additional functional as well as molecular assays should be employed to do so. However, these data include detailed information about the clinical features of acute myelomonocytic leukemia and will help researchers to evaluate and understand the effects of new therapeutic agents.

Acute myeloid leukemia (AML) is characterized by the uncontrolled proliferation of hematopoietic myeloid cells that are blocked at different stages of maturation. This dysregulation can affect the granulocytic, monocytic, erythrocytic or megaryocytic differentiation pathways1. AML cells accumulate in the bone marrow, leading to impaired hematopoiesis, which results in thrombopenia, lymphopenia and anemia. The leukemic cells also invade the blood and non-lymphoid organs.
The C1498 mouse model has been used for decades as a model for acute leukemia since cancer cells were isolated from a leukemic 10-month-old C57BL/6 (H-2b) female mouse in 1941. The literature describes the invasion into the blood, hematopoietic organs (e.g., the spleen and lymph nodes) and non-hematopoietic organs (e.g., the liver, lungs, ovaries, and kidneys) by highly proliferative C1498 cells after they were injected via an intravenous, subcutaneous or intra-peritoneal route into susceptible mice2-4. However, this mouse model was reported to induce either granulocytic2,5 or myelomonocytic6 leukemia. More recently, a study published in 2002 described this type of cancer as murine NKT cell leukemia7. Thus, the literature differs concerning the nature of this C1498 cell line and the associated leukemia it induces in mice. These discrepancies are mainly due to a lack of detailed and updated published information about the cells and the leukemic disease in general because many studies were performed in the 1950 - 70&#39;s.
Here we provide a detailed protocol to describe how to characterize C1498 cells and analyze the nature of the leukemic disease that is induced by their intravenous injection into mice. The first section of this protocol is dedicated to a description of C1498 cells that have been cultured in vitro. Fluorescent antibodies directed against surface and intracellular hematopoietic markers were used to determine their phenotype using flow cytometry. The presence of myeloperoxidase was assessed using immunofluorescence microscopy, their hematopoietic lineage and differentiation stage were evaluated using cytochemistry to assess the activity of esterases, and May-Gr&#252;nwald Giemsa staining was performed. The C1498 cells were then injected into mice, and the acute leukemia disease that was induced is described in the second section of this manuscript. The same techniques were used to determine the frequencies and the phenotypes of leukemic and inherent cells in the bone marrow, peripheral blood, spleen and non-hematopoietic organs (the liver and lungs).
This protocol is highly reproducible, and the data presented here will help researchers to assess the effects of new therapeutic strategies. This leukemia mouse model has already been used to test immunotherapy approaches and different cancer chemotherapeutic drugs8,9. Their efficacy was evaluated by determining the evolution of tumor burden and survival rates. This protocol can be used to provide additional information about the distribution and subsistence of leukemic and other hematopoietic cell populations during treatment.

<table><tbody><tr><td>C1498 cell line</td><td>ATCC</td><td>TIB 49</td><td></td></tr><tr><td>C57BL/6J-Ly5.1</td><td>Charles River</td><td>B6.SJL-Ptprc a Pep3 b/BoyCrl</td><td></td></tr><tr><td>&lt;strong&gt;Cells culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol, Gibco</td><td>ThermoFisher Scientific</td><td>21985</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher Scientific</td><td>10270</td><td></td></tr><tr><td>HEPES, Gibco (1 M)</td><td>ThermoFisher Scientific</td><td>15630</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution, Gibco&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11140</td><td></td></tr><tr><td>Penicillin-Streptomycin, Gibco&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15140</td><td></td></tr><tr><td>RPMI 1640 Medium (Gibco, GlutaMAX Supplement)</td><td>ThermoFisher Scientific</td><td>61870</td><td></td></tr><tr><td>Sodium Pyruvate, Gibco (100 mM)</td><td>ThermoFisher Scientific</td><td>11360</td><td></td></tr><tr><td>&lt;strong&gt;Flow cytometry staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse B220 APC (1)</td><td>ebiosciences</td><td>17-0452</td><td>clone RA3-6B2, final dilution 1/100</td></tr><tr><td>anti-mouse B220 biotin (2)</td><td>ebiosciences</td><td>13-0452</td><td>clone RA3-6B2, 1/400</td></tr><tr><td>anti-mouse CD3 eFluor450 (1)</td><td>ebiosciences</td><td>48-0032</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE (2)</td><td>BD biosciences</td><td>555275</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE-Cy5 (3)</td><td>ebiosciences</td><td>15-0031</td><td>clone 145-2C11, 1/100</td></tr><tr><td>anti-mouse CD4 APC (1)</td><td>ebiosciences</td><td>17-0041</td><td>clone GK1.5, 1/500</td></tr><tr><td>anti-mouse CD4 PE (2)</td><td>ebiosciences</td><td>12-0041</td><td>clone GK1.5, 1/200</td></tr><tr><td>anti-mouse CD8 biotin (1)</td><td>ebiosciences</td><td>13-0081</td><td>clone 53-6.7, 1/100</td></tr><tr><td>anti-mouse CD8 eFluor450 (2)</td><td>ebiosciences</td><td>48-0081</td><td>clone 53-6.7, 1/500</td></tr><tr><td>anti-mouse CD11a biotin</td><td>ebiosciences</td><td>13-0111</td><td>clone M17/4, 1/100</td></tr><tr><td>anti-mouse CD11b PE&amp;nbsp;</td><td>ebiosciences</td><td>12-0112</td><td>clone M1/70, 1/200</td></tr><tr><td>anti-mouse CD16/32 biotin</td><td>ebiosciences</td><td>13-0161</td><td>clone 93, 1/400</td></tr><tr><td>purified anti-mouse CD16/32 (FcR blocking)&amp;nbsp;</td><td>BD biosciences</td><td>553141</td><td>clone 2.4G2</td></tr><tr><td>anti-mouse CD18 biotin (1)</td><td>ebiosciences</td><td>13-0181</td><td>clone M18/2, 1/100</td></tr><tr><td>anti-mouse CD18 FITC (2)</td><td>ebiosciences</td><td>11-0181</td><td>clone M18/2, 1/50</td></tr><tr><td>anti-mouse CD19 PE</td><td>ebiosciences</td><td>12-0193</td><td>clone 1D3, 1/200</td></tr><tr><td>anti-mouse CD21/35 PE</td><td>ebiosciences</td><td>12-0211</td><td>clone 8D9, 1/50</td></tr><tr><td>anti-mouse CD31 PE</td><td>ebiosciences</td><td>12-0311</td><td>clone 390, 1/100</td></tr><tr><td>anti-mouse CD34 eFluor660</td><td>ebiosciences</td><td>50-0341</td><td>clone RAM34, 1/20</td></tr><tr><td>anti-mouse CD44 PE</td><td>BD biosciences</td><td>553134</td><td>clone IM7, 1/50</td></tr><tr><td>anti-mouse CD45.2 FITC&amp;nbsp;</td><td>ebiosciences</td><td>11-0454</td><td>clone 104, 1/100</td></tr><tr><td>anti-mouse CD49b/Pan NK PE</td><td>&amp;nbsp;BD biosciences</td><td>553858</td><td>clone DX5, 1/50</td></tr><tr><td>anti-mouse CD80 biotin</td><td>ebiosciences</td><td>13-0801</td><td>clone 16-10A1, 1/200</td></tr><tr><td>anti-mouse CD86 biotin</td><td>ebiosciences</td><td>13-0862</td><td>clone GL1, 1/200</td></tr><tr><td>anti-mouse CD107b (Mac-3) PE</td><td>ebiosciences</td><td>12-5989</td><td>clone M3/84, 1/40</td></tr><tr><td>anti-mouse CD115 PE</td><td>ebiosciences</td><td>12-1152</td><td>clone AFS98, 1/100</td></tr><tr><td>anti-mouse CD117 eFluor450</td><td>ebiosciences</td><td>48-1171</td><td>clone 2B8, 1/100</td></tr><tr><td>anti-mouse CD127 PE</td><td>ebiosciences</td><td>12-1271</td><td>clone A7R34, 1/100</td></tr><tr><td>anti-mouse CD150 APC</td><td>ebiosciences</td><td>17-1501</td><td>clone 9D1, 1/20</td></tr><tr><td>anti-mouse CD274 (PD-L1) biotin</td><td>ebiosciences</td><td>13-5982</td><td>clone MIH5, 1/200</td></tr><tr><td>anti-mouse Ly-6A/E (Sca-1) PE</td><td>ebiosciences</td><td>12-5981</td><td>clone D7, 1/100</td></tr><tr><td>anti-mouse Ly-6C APC</td><td>ebiosciences</td><td>17-5932</td><td>clone HK1.4, 1/200</td></tr><tr><td>anti-mouse Ly-6G&amp;nbsp; (Gr-1) biotin (1)</td><td>ebiosciences</td><td>13-5931</td><td>clone RB6-8C5, 1/400</td></tr><tr><td>anti-mouse Ly-6G FITC (2)</td><td>ebiosciences</td><td>11-9668</td><td>clone 1A8, 1/50</td></tr><tr><td>anti-mouse MHC class I (H-2Db) biotin</td><td>ebiosciences</td><td>13-5999</td><td>clone 28-14-8, 1/50</td></tr><tr><td>anti-mouse MHC class II (I-A/I-E) PE-Cy5</td><td>ebiosciences</td><td>15-5321</td><td>clone M5/114.15.2, 1/1000</td></tr><tr><td>anti-mouse NK1.1 PE</td><td>BD biosciences</td><td>557391</td><td>clone PK136, 1/50</td></tr><tr><td>anti-mouse TCRVb FITC</td><td>BD biosciences</td><td>553170</td><td>clone H57-597, 1/50</td></tr><tr><td>streptavidin PE-Cy5</td><td>ebiosciences</td><td>15-4317</td><td>&amp;nbsp;&amp;nbsp; 1/200</td></tr><tr><td>Immunofluorescence staining reagents</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse myeloperoxidase (heavy chain antibody)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>sc-16129</td><td>1/10 (20 &amp;micro;g/ml)</td></tr><tr><td>&amp;nbsp;anti-goat IgG (Texas Red coupled antibody)</td><td>Jackson Immunoresearch</td><td>705-076-147</td><td>1/250</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immunoresearch</td><td>017-000-001</td><td></td></tr><tr><td>Hoechst solution</td><td>BD Biosciences</td><td>561908</td><td>1/1000</td></tr><tr><td>Mounting medium 1 (Fluoromount)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4680</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>&lt;strong&gt;Esterase cytochemical staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Naphtol AS-D chloroacetate solution</td><td>Sigma-Aldrich</td><td>911</td><td></td></tr><tr><td>Dye solution (Fast Red Violet LB Base solution)</td><td>Sigma-Aldrich</td><td>912</td><td></td></tr><tr><td>pH6.3 buffer concentrate (TRIZMAL)</td><td>Sigma-Aldrich</td><td>913</td><td></td></tr><tr><td>Sodium nitrite solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Sigma-Aldrich</td><td>GHS3</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Formaldehyde solution, 37%</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>&amp;alpha;-naphthyl butyrate solution</td><td>Sigma-Aldrich</td><td>1801</td><td></td></tr><tr><td>Phosphate buffer solution</td><td>Sigma-Aldrich</td><td>1805</td><td></td></tr><tr><td>Sodium nitrite Tablet</td><td>Sigma-Aldrich</td><td>1809</td><td></td></tr><tr><td>Pararosaniline solution</td><td>Sigma-Aldrich</td><td>1804</td><td></td></tr><tr><td>Methylene blue solution</td><td>Sigma-Aldrich</td><td>1808</td><td>1/10</td></tr><tr><td>mounting medium 2 (Clearmount)</td><td>Invitrogen</td><td>00-8010</td><td></td></tr><tr><td>&lt;strong&gt;May-Gr&amp;uuml;nwald Giemsa staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald solution</td><td>RAL Diagnostics</td><td>320070</td><td></td></tr><tr><td>Giemsa R solution&amp;nbsp;</td><td>RAL Diagnostics</td><td>320310</td><td>&amp;nbsp;&amp;nbsp; 1/20</td></tr><tr><td>pH 6.8 buffer solution</td><td>RAL Diagnostics</td><td>330368</td><td></td></tr><tr><td>&lt;strong&gt;Others materials, reagents and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamine 1000 (100 mg/ml)</td><td>VIRBAC</td><td>3597132111010</td><td></td></tr><tr><td>Xylazine SEDAXYLAN (20 mg/ml)</td><td>CEVA</td><td></td><td></td></tr><tr><td>Bovin albumin serum (BSA) powder</td><td>ThermoFisher Scientific</td><td>BP671</td><td></td></tr><tr><td>PBS solution (1x concentrate)</td><td>ThermoFisher Scientific</td><td>14190</td><td></td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Ultra Pure 0.5 M EDTA solution, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575</td><td></td></tr><tr><td>Separating solution (Pancoll Mouse)</td><td>PANBIOTECH</td><td>P04-64100</td><td></td></tr><tr><td>Lysis buffer (Red Blood Cells Lysing Buffer) (10x)</td><td>BD Biosciences</td><td>555899</td><td>1/10</td></tr><tr><td>NaOH 10 N</td><td>ThermoFisher Scientific</td><td>SS267</td><td></td></tr><tr><td>Trypan blue solution (0.4 %)</td><td>ThermoFisher Scientific</td><td>15250-061</td><td>1/2</td></tr><tr><td>50 ml tube (Falcon)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer (Falcon)&amp;nbsp;</td><td>Corning Life Sciences</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;Chamber &amp;amp; filter card (EZ Cytofunnel Shandon)</td><td>Thermo Scientific</td><td>A78710003</td><td></td></tr><tr><td>Microscope Cover Glasses, 24 x 24mm</td><td>Knittel Glass</td><td>VD1 2424 Y100</td><td></td></tr><tr><td>Slides (Starfrost - ground edges 90)</td><td>Knittel Glass</td><td>VS1137# 077FKB</td><td></td></tr><tr><td>EDTA Tube</td><td>Greiner Bio-One</td><td>454034</td><td></td></tr><tr><td>Pasteur Pipette 150 mm (capillary tube)</td><td>Fisherbrand</td><td>1154-6963</td><td></td></tr><tr><td>26 G needle</td><td>Terumo</td><td>NN-2613R</td><td></td></tr><tr><td>&amp;nbsp;insulin syringe and needle 29 G</td><td>Terumo</td><td>BS05M2913</td><td></td></tr><tr><td>30 G needle&amp;nbsp;</td><td>Becton &amp;amp; Dickinson</td><td>304000</td><td></td></tr><tr><td>Flow cytometry tubes (blue)</td><td>Beckman Coulter</td><td>2523749</td><td></td></tr><tr><td>Water-repellent pen (Dakopen)</td><td>Dako</td><td>S200230</td><td></td></tr><tr><td>Sharp sterile scissors&amp;nbsp;</td><td>Nessi-care</td><td>SCI-01</td><td></td></tr><tr><td>Sterile forceps</td><td>Dominique Dutscher</td><td>956506</td><td></td></tr><tr><td>Thoma cell counting chamber</td><td>VWR</td><td>631-0397</td><td></td></tr><tr><td>Petri Dishes (Fisherbrand Plastic)</td><td>ThermoFisher Scientific</td><td>S33580A</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 ml)</td><td>ThermoFisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Cylindrical Restrainer 15 - 30 gm</td><td>Stoelting</td><td>51338</td><td></td></tr><tr><td>Shandon Cytospin 3 Cytocentrifuge</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SS-10L</td><td></td></tr><tr><td>1 ml syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.08543</td><td></td></tr><tr><td>Sterile gauze sponges</td><td>URGO</td><td>501580</td><td></td></tr></tbody></table>

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

To characterize the C1498 mouse model, we proceeded with two major steps. First, the C1498 cells were characterized to determine their hematopoietic lineage and maturation stage in vitro (Figure 1). These cells were then injected into congenic mice, and the nature of the induced leukemic disease was assessed to determine different features: leukemic cell infiltration, their phenotype, a quantification of the hematopoietic cells (mature and progenitors/precursors) in bone marrow, the frequencies of C1498 cells and mature hematopoietic cells in the blood and an evaluation of organ swelling (in the spleen, liver, and lungs) and cellular composition.
To characterize the C1498 cell phenotypes in vitro, the cells were labeled with antibodies directed against molecules that are expressed by hematopoietic precursors and mature cells (Table 1), and the results were analyzed using flow cytometry. The C1498 cells were positive for cell surface expression of Mac-1 (CD11b/CD18) (&#8764; 7%), B220 (&#62; 25%), and they displayed intracellular expression of CD3&#949;, T-Cell Receptor (TCR) V&#946; chains and Mac-3 (Figures 2A and B). The cells were negative for the cell surface markers Ly6G, Ly6C, CD115, CD21/CD35, CD19, CD3, CD4, CD8, NK1.1, and pan-NK molecules and for the intracellular expression of CD4 and CD8 (data not shown). They were then examined for markers of hematopoietic stem cells and progenitors (Table 1). They were also negative for cell surface expression of CD117, CD34, Sca-1, CD150 and CD16/32 (data not shown). These leukemic cells were then tested to determine the expression of adhesion, antigen presentation and co-stimulatory molecules. The cells expressed the surface markers LFA-1 (CD11a/CD18), CD44, CD31 (PECAM-1), and H-2Db and were negative for MHC class II, CD80, CD86 and CD274 (data not shown). C1498 cells therefore expressed both myeloid (Mac-1, Mac-3) and lymphoid markers (B220, CD3, TCR).
To better characterize their hematopoietic lineage, myeloperoxidase expression was assessed using immunofluorescence microscopy. All of the cells were positive for the myeloperoxidase, which verified their myeloid origin (Figure 3A). A majority of the cells also stained positive for &#945;-naphthyl butyrate esterases (Figure 3B, left panel), and some of them stained for the naphthol AS-D chloroacetate esterases (black arrows) (Figure 3B, right panel). The results indicate that the cells contained mixtures of monocytic and granulocytic cells. After May-Gr&#252;nwald Giemsa staining was performed, the C1498 cells were observed to display a blast-like morphology with a high nucleo-cytoplasmic ratio, 3 to 5 nucleoli in the nucleus, a perinuclear halo, numerous vacuoles and a basophilic cytoplasm (Figure 3C). Thus, the C1498 cell line is composed of monoblasts and myeloblasts.
The C1498 cells (CD45.2+) were then intravenously injected into CD45.1+ mice. The mice succumbed 17 to 19 days after the cells were injected. These mice were sacrificed so that their leukemia type could be analyzed before they died from the disease. The control mice, which were injected with PBS, were analyzed at the same time points for comparison. The C1498 cell-injected mice displayed massive infiltration of C1498 cells into their bone marrow, as demonstrated by the blast-like appearance of the cells after May-Gr&#252;nwald Giemsa staining was performed (Figure 4A). They also preserved their monocytic and granulocytic phenotypes (Figure 4B and C), demonstrating an accumulation of monoblastic and myeloblastic cells that is characteristic of acute myelomonocytic leukemia.
To determine whether medullary hematopoietic cells numbers were lower following leukemic cells invasion, CD45.2+ C1498 cells, B lymphocytic, monocytic and granulocytic populations (including progenitors, precursors and mature cells), were quantified using immunofluorescent staining and multi-parametric flow cytometry analysis. Leukemic cells represented 16 to 36% of the hematopoietic cells (data not shown). The other cell types were all present in significantly lower numbers in the C1498-injected mice than in the PBS-injected mice (by 5-fold on average for B cell subsets, 4-fold on average for granulocytic cells and 3-fold on average for monocytic subsets) (Figure 5A to C).
An investigation of the frequencies of mononuclear cells in leukemic and control mouse blood samples showed that they contained a comparable percentage of lymphocytes (Figure 6A) but a higher frequency of monocytic and leukemic cells. These characteristics are representative of acute myelomonocytic leukemia11 (Figure 6B).
Among the other features of acute myelomonocytic leukemia12, the C1498-injected mice presented with swollen livers (hepatomegaly), lungs and spleens (splenomegaly) (Figure 7A). Various frequencies of CD45.2+ C1498 cells were detected in these organs using immunofluorescent staining and flow cytometry analysis (Figure 7B). As splenomegaly can result from high numbers of infiltrated monocytes, we also estimated the proportions of splenic populations. The numbers of cells in the B lymphocytic, monocytic and granulocytic cell fractions were significantly larger, by an average of 2-fold, 2.5-fold and 3-fold, respectively, in leukemic spleens than in control spleens (Figure 7C).
<img alt="Figure 1" src="/files/ftp_upload/54270/54270fig1.jpg" /> 
Figure 1. Schematic Representation of the Protocol Set Up for Characterizing in vitro Cultured C1498 Cell Lines and in vivo Descriptions of Acute Leukemia. The hematopoietic lineage and the differentiation stage of tissue-cultured C1498 cells were first determined. C1498 cells were then injected into congenic mice to induce the development of acute leukemia. The isolation of bone marrow, peripheral blood, spleen, liver and lung tissues was performed to determine the frequencies, phenotypes and morphological changes after the C1498 cells infiltration. IV: Intravenous MGG: May-Gr&#252;nwald Giemsa. <a href="http://cloudflare.jove.com/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54270/54270fig2.jpg" /> 
Figure 2. Phenotypic Analysis of C1498 Cells after in vitro Culture. Representative flow cytometry dot plots and histograms of cell surface (A) and intracellular (B) C1498-expressed molecules that were associated with hematopoietic mature cell differentiation are shown. C1498 cells were harvested from cultures, washed and labeled using fluorescent antibodies that were specific for the cell surface CD11b, CD18 and B220 markers or their isotype controls. For intracellular staining, the cells were fixed, permeabilized and labeled using antibodies directed against Mac-3, CD3&#949;, and a common epitope of the TCR (T-Cell Receptor) V&#946; chain or their isotype controls. Analyses were performed using gating with live cells. <a href="http://cloudflare.jove.com/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54270/54270fig3.jpg" /> 
Figure 3. Functional and Morphological Characterization of Cultured C1498 Cells. C1498 cells were harvested from cultures and centrifuged on slides for microscopy. (A) Staining for myeloperoxidase expression was performed using immunofluorescence. (B) Cytochemical reactions were used to analyze the &#945;-naphthyl butyrate esterase (NBE) and naphthol AS-D chloroacetate esterase (CAE) activities in C1498 cells. Cells were considered to be positive for each label when brown and red-purple, large cytoplasmic granules, respectively, were observed. (C) May-Gr&#252;nwald Giemsa (MGG) staining of C1498 cells. For each staining experiment, the microscopy objective magnification is indicated. Each image is representative of three separate experiments. <a href="http://cloudflare.jove.com/files/ftp_upload/54270/54270fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54270/54270fig4.jpg" /> 
Figure 4. Bone Marrow Morphologies in PBS- and C1498-injected Mice. Bone marrow cells were isolated from PBS- and C1498 cell-injected mice and centrifuged onto slides for microscopy. (A) May-Gr&#252;nwald Giemsa (MGG) staining. (B) &#945;-naphthyl butyrate esterase (NBE) and (C) naphthol AS-D chloroacetate esterase (CAE) functions were evaluated using cytochemistry. In panel A, the band (immature) or segmented (mature) neutrophils are less visible in the bone marrow of the C1498-injected mice than the PBS-injected mice. Panel B and C indicate that there was an accumulation of monocytic and granulocytic cells in the leukemic bone marrow compared to the numbers observed in the control bone marrow. All microscopic analyses were performed using a 100X magnification objective. <a href="http://cloudflare.jove.com/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54270/54270fig5.jpg" /> 
Figure 5. Quantitative Analysis of Medullary Populations in PBS- and C1498-injected Mice. Bone marrow cells were isolated from PBS- and C1498 cell-injected mice and estimated after cell counting was performed. The frequencies of the different cell populations were determined after immunostaining and live cell gated flow cytometry analysis. (A) The B cell subsets included CD19+B220+cells in stages from pro-B cells to mature B lymphocytes (B) granulocytic cells in the CD3- and CD11b+Ly6G+ lineages, which included precursors and immature and mature granulocytes. (C) The monocytic subsets were defined as CD3-CD115+ and included cells in the progenitor to mature monocyte stages. n = 7 mice/group, and the data are presented as histograms showing the means &#177; SEM. ***, p&#60; 0.0001 and **, p&#60; 0.01, unpaired Student&#39;s t-test comparing PBS- and C1498-injected mice. <a href="http://cloudflare.jove.com/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54270/54270fig6.jpg" /> 
Figure 6. Blood Analysis of Mononuclear Cell Subsets in PBS- and C1498-injected mice. Representative flow cytometry dot plots of (A) T and B lymphocyte percentages, which were respectively defined as CD3+ and B220+ cells in PBS- and C1498 cell-injected mice. (B) Monocytic cell frequencies in C1498 leukemic and control (PBS) mice were determined by analyzing CD115+Ly6C- and CD115+Ly6Chigh cells. The analysis was performed by gating live cells. To compare leukemic and control mice, CD45.2+ C1498 cells were excluded. <a href="http://cloudflare.jove.com/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54270/54270fig7.jpg" /> 
Figure 7. Estimation of Splenic Populations in Leukemic and Control Mice. (A) Representative photographs of liver, lung and spleen swelling in leukemic mice compared to control mice. Spleens were collected and weighed, and splenocytes were counted following tissue disruption. (B) Histogram representing leukemic cell frequencies in different organs after immunostaining was performed for CD45.2+ cells and the results were analyzed using flow cytometry. (C) Estimations of splenic B, granulocytic and monocytic cell numbers after immunostaining and flow cytometry analysis gating were performed to identify live CD19+B220+, CD3-CD11b+Ly6G+, CD3-CD11b+Ly6C- and CD3-CD11b+Ly6Chigh cells. The scale bars shown for the lungs, spleens and livers indicate 1 cm. n = 5 - 8 mice/group, and the data are represented in histograms as the means&#160;&#177; SEM. *, p&#60; 0.05; **, p= 0.0033, unpaired Student&#39;s t-test comparing PBS- and C1498-injected mice. <a href="http://cloudflare.jove.com/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Cell Type</td>
			<td>Membrane or Intracellular Molecules&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precursors and Mature cells</td>
			<td></td>
		</tr>
		<tr>
			<td>NK cells</td>
			<td>NK1.1+, pan-NK+</td>
		</tr>
		<tr>
			<td>NKT cells</td>
			<td>NK1.1+, pan-NK+, TCR Vbeta+(8.2), CD3+</td>
		</tr>
		<tr>
			<td>T lymphocytes</td>
			<td>TCR Vbeta+, CD3+, CD4+, CD8+</td>
		</tr>
		<tr>
			<td>B cells precursors and B lymphocytes</td>
			<td>B220+, CD19+, CD21/35+&#160;</td>
		</tr>
		<tr>
			<td>granulocytic precursors and granulocytes</td>
			<td>Ly6G+, Mac-1+, CD11b+</td>
		</tr>
		<tr>
			<td>monocytic precursors and monocytes/macrophages</td>
			<td>CD11b+, Mac-1+, Mac-3+, CD21/35+, CD115+, Ly6Chi</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Progenitors</td>
			<td></td>
		</tr>
		<tr>
			<td>multipotent progenitors</td>
			<td>CD117+ Sca-1+ CD34+ (Lin- CD150-)</td>
		</tr>
		<tr>
			<td>lymphoid-primed multipotent progenitors&#160;</td>
			<td>CD117hi Sca-1hi CD127+ (Lin- )</td>
		</tr>
		<tr>
			<td>common lymphoid progenitors&#160;</td>
			<td>CD117lo Sca-1lo CD127+ (Lin- )</td>
		</tr>
		<tr>
			<td>common myeloid progenitors&#160;</td>
			<td>CD16/32lo CD117+ CD34int (Lin- Sca-1-)</td>
		</tr>
		<tr>
			<td>granulocyte-macrophage progenitors&#160;</td>
			<td>CD16/32hi CD117+ CD34hi (Lin- Sca-1-)</td>
		</tr>
		<tr>
			<td>megakaryocyte-erythroid progenitors&#160;</td>
			<td>CD16/32lo CD117+ CD34lo (Lin- Sca-1-)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematopoietic stem cells&#160;</td>
			<td>CD117+ Sca-1+ CD150+ (Lin- CD34-)</td>
		</tr>
	</tbody>
</table>
Table 1. Markers of Hematopoietic Cell Lineages and Differentiation. 
CD: cluster of differentiation; Lin: markers of mature cells; lo: low expression; hi: high expression; int: intermediate expression; NK: natural killer cells; TCR: T-cell receptor.

Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

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19.9K Views.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC). This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Grünwald Giemsa staining.

Video: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

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Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

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Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

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Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

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Medicine

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

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Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

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Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

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Immunology and Infection

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

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

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