This work was supported by The Fox Chase Cancer Center Board of Directors (DDM), the American Society of Hematology Scholar Award (SMS), American Cancer Society RSG (SMS) and the Department of Defense (Award#: W81XWH-18-1-0472).

All of the animal procedures described in this protocol have been approved by the Institutional Animal Care and Use committee (IACUC) at Fox Chase Cancer Center.
NOTE: The protocol workflow is divided into 4 parts as presented in Figure 1 and the required reagents are listed in the Table of Materials.
1. Bone Marrow (BM) Isolation
NOTE: MLL-AF9 leukemia mice were generated as described previously11.
<ol>
	<li>Recover mono-nuclear bone marrow cells, as described previously12,13,14,15, from wild type C57.Bl6 mice (which express the CD45.2 congenic marker) as well as from C57.Bl6-SJL mice ( which express the CD45.1 congenic marker) that have been transplanted with MLL-AF9-expressing leukemia cells (CD45.2+). 
	NOTE: BM can be recovered from mice either by crushing12,13 or by flushing bones14,15. For the experiments presented here, BM was recovered from both healthy and leukemia mice via flushing.</li>
</ol>
2. Mitochondrial ROS Fluorogenic Dye Staining
<ol>
	<li>Once mono-nuclear bone marrow cells have been recovered from healthy and/or leukemia mice, stain an aliquot of cells with Trypan Blue and count using a hemocytometer to determine the starting number of total BM cells.</li>
	<li>Centrifuge the cells at 300 x g for 5 min. Aspirate the supernatant and resuspend the pellet in F-PBS (PBS supplemented with 2% fetal bovine serum and a 1% Penicillin/Streptomycin cocktail) to a concentration of 2 x 106 cells/mL.</li>
	<li>Aliquot 200 &#181;L of cell suspension per tube into 9 single-color control tubes labeled as follows: 
	No stain, B220-Cy5-PE, cKit-Cy7-APC, Sca1-PacBlue, CD150-APC (for healthy HSPCs only), CD45.2-APC (for leukemia cells only), CD34-FITC, Mitochondrial ROS dye and Live/dead cell stain. 
	NOTE:&#160;(Optional) A positive control for the induction of mitochondrial ROS can be prepared by treating 2 x 105 cells in 200 &#181;L with 20 &#181;M of Menadione Sodium Bisulfite (MSB) for 1 h at 37 &#730;C in a 5% CO2 incubator. A second control to reverse the MSB-mediated induction of mitochondrial ROS can be prepared by treating 2 x 105 cells in 200 &#181;L with 20 &#181;M of MSB plus 100 &#181;M N-acetyl-L-cysteine (NAC) for 1 h at 37 &#730;C in a 5% CO2 incubator.</li>
	<li>Aliquot the remaining cells in a tube (experimental tube) and centrifuge at 300 x g for 5 min.</li>
	<li>Resuspend the cells in F-PBS with a live/dead cell stain according to the manufacturer&#8217;s instructions. Incubate on ice for 30 min. Be sure to add live/dead stain to the single-color control tube.</li>
	<li>Add 1.0 mL of room temperature (RT) F-PBS to both single-color and experimental tubes stained with the live/dead dye. Centrifuge 5 min at 300 x g at RT.</li>
	<li>Resuspend 50 &#181;g of the mitochondrial ROS dye in 13 &#181;L of dimethyl sulfoxide (DMSO) to obtain a 5 mM stock solution.</li>
	<li>Dilute mitochondrial ROS dye to a final concentration of 5 &#181;M in RT F-PBS with or without Verapamil (50 &#181;M).</li>
	<li>Aspirate off the wash of the live/dead cell stain. Add 200 &#181;L of mitochondrial ROS dye stain containing Verapamil to each experimental tube as well as the mitochondrial ROS dye single-color control tube.</li>
	<li>Vortex to mix and incubate for 10 min at 37 &#730;C in the dark.</li>
	<li>Add 1.0 mL of RT F-PBS to the mitochondrial ROS-stained single-color control and experimental tubes. Centrifuge 5 min at 300 x g at RT.</li>
	<li>Aspirate off the supernatant and wash the cells with an additional 1.0 mL of RT F-PBS. Centrifuge 5 min at 300 x g at RT.</li>
</ol>
3. Lineage Antibody Staining
<ol>
	<li>Prepare the antibody cocktails listed in Table 1. 
	NOTE: These antibodies cocktails have been optimized previously14,15,16.</li>
	<li>Aspirate the supernatant from the final mitochondrial ROS dye wash of the experimental tubes containing healthy BM and add 200 &#181;L of antibody cocktail #1 to each tube. Vortex to mix. Also prepare the single-color control tubes. Incubate for 60 min on ice in the dark.</li>
	<li>Aspirate the supernatant from the final mitochondrial ROS dye wash of the experimental tubes containing leukemia BM and add 200 &#181;L of the antibody cocktail #2 to each tube. Vortex to mix. Incubate for 60 min on ice in the dark.</li>
	<li>Wash with 1.0 mL of cold F-PBS and centrifuge 5 min at 300 x g at RT.</li>
	<li>Resuspend cells in 500 &#181;L of cold F-PBS and filter the cells in a flow cytometer tube using a 40 &#181;m filter to exclude aggregates.</li>
</ol>
4. Flow Cytometry Acquisition and Analysis
NOTE: Several hematopoietic stem and progenitor subsets are rare, such as long-term hematopoietic stem cells. Thus, ideally 3-5 million events should be collected for each experimental tube during flow cytometry acquisition for sufficient analysis of mitochondrial ROS in the various HSPC subsets.
<ol>
	<li>Use the no-stain control tube to set the forward (FSC-A) and side (SSC-A) scatter plots based on the size and complexity of the cell population analyzed.</li>
	<li>Use the no-stain and single-color control tubes to compensate the flow cytometer.</li>
	<li>Gate out extraneous debris from the forward and side scatter plot (Figure 2A,B, first panel from the left).</li>
	<li>Gate out doublets using a double discriminator such as the forward discriminator (Figure 2A,B, second panel from the left).</li>
	<li>Follow the gating strategy proposed in Figure 2A,B to select live cells, lineage low cells and the various HSPC and leukemia subsets.</li>
	<li>For each population of interest, analyze the median fluorescence intensity (MFI) of the TRPE channel (x-axis) in a histogram plot to evaluate differences in the mitochondrial ROS signal (Figure 3A-C, left panels). Levels of mitochondrial ROS can be evaluated at the single-cell level by comparing mitochondrial ROS staining versus specific lineage markers in a scatter plot.</li>
</ol>

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The authors have nothing to disclose.

Fluorogenic dyes that have been developed for the detection of ROS are frequently evaluated in fixed cells by microscopy or in live cells by flow cytometry22. Flow cytometric evaluation of mitochondrial ROS in BM cells using mitochondrial ROS fluorogenic dyes has two major advantages: 1) It is a fast and simple technique that is suitable for live cell analysis and 2) it allows for distinguishing and analyzing rare populations at the single-cell level in the BM using surface marker staining. The st...

Reactive Oxygen Species (ROS) are highly reactive molecules derived from molecular oxygen. The most well-defined cellular location of ROS production is the mitochondria, where electrons that pass through the electron transport chain (ETC) during oxidative phosphorylation (OXPHOS) are absorbed by molecular oxygen leading to the formation of a specific type of ROS called superoxides1. Through the actions of a series of enzymes, called superoxide dismutases or SODs, superoxides are converted into hydrogen peroxides, which are subsequently neutralized into water by enzymes such as catalase or glutathione peroxidases (GPX). Perturbations in ROS-regulatory mechanisms can lead to the excess production of ROS, often referred to as oxidative stress, which have harmful and potentially lethal cellular consequences such as macromolecule damage (i.e., DNA, protein, lipids). Moreover, oxidative stress is related to several pathologies, such as diabetes, inflammatory diseases, aging and tumors2,3,4. To maintain redox homeostasis and prevent oxidative stress, cells possess a variety of ROS-regulating mechanisms5.
Physiological levels of certain ROS are necessary for proper embryonic and adult hematopoiesis6. However, excess ROS is associated with DNA damage, cellular differentiation and exhaustion of the hematopoietic stem and progenitor pool. There is also evidence that alterations in redox biology may differ between leukemia and healthy cells. For example, ROS levels tend to be higher in acute myeloid leukemia (AML) cells relative to their healthy counterparts and other studies have suggested that leukemia stem cells maintain a low steady-state level of ROS for survival7,8. Importantly, strategies for therapeutically capitalizing on these redox differences have shown promise in several human cancer settings9,10. Therefore, assays that allow for the assessment of ROS levels in mouse models may improve our understanding of how these species contribute to cellular physiology and disease pathogenesis as well as potentially provide a platform for assessing the effectiveness of novel redox-targeting anti-cancer therapies.

<table><tbody><tr><td>Heat inactivated FBS</td><td>VWR Seradigm LIFE SCIENCE</td><td>97068-085</td><td>Media</td></tr><tr><td>Penicillin Streptomycin</td><td>Corning</td><td>30-002-CI</td><td>Media</td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>BP399-20</td><td>Buffer</td></tr><tr><td>15 mL conical tube</td><td>BD falcon</td><td>352096</td><td>Tissue Culture Supplies</td></tr><tr><td>50 mL conical tube</td><td>BD falcon</td><td>352098</td><td>Tissue Culture Supplies</td></tr><tr><td>40 &amp;mu;m cell strainers</td><td>Fisher Scientific</td><td>22-363-547</td><td>Tissue Culture Supplies</td></tr><tr><td>RBC Lysis Buffer</td><td>Fisher Scientific</td><td>50-112-9751</td><td>Tissue Culture Supplies</td></tr><tr><td>Menadione sodium Bisulfite</td><td>Sigma aldrich</td><td>M5750</td><td>Pro-oxidant</td></tr><tr><td>NAC</td><td>Sigma aldrich</td><td>A7250</td><td>Anti-oxidant</td></tr><tr><td>CD3 PE-Cy5 clone 145-2c11</td><td>Biolegend</td><td>100310</td><td>Antibody</td></tr><tr><td>CD4 PE-Cy5 clone RM4-5</td><td>eBioscience</td><td>15-0041-81</td><td>Antibody</td></tr><tr><td>CD8 PE-Cy5 clone 53-6.7</td><td>eBioscience</td><td>15-0081-81</td><td>Antibody</td></tr><tr><td>CD19 PE-Cy5 clone 6D5</td><td>Biolegend</td><td>115510</td><td>Antibody</td></tr><tr><td>B220 PE-Cy5 clone RA3-6B2</td><td>Biolegend</td><td>103210</td><td>Antibody</td></tr><tr><td>Gr1 PE-Cy5 clone RB6-8C5</td><td>Biolegend</td><td>108410</td><td>Antibody</td></tr><tr><td>Ter119 PE-Cy5 clone Ter-119</td><td>Biolegend</td><td>116210</td><td>Antibody</td></tr><tr><td>CD48 PE-Cy5 clone HM48-1</td><td>Biolegend</td><td>103420</td><td>Antibody</td></tr><tr><td>CD117&amp;nbsp; APC-Cy7 clone 2B8</td><td>Biolegend</td><td>105825</td><td>Antibody</td></tr><tr><td>Sca1 peacific Blue clone D7</td><td>Biolegend</td><td>108120</td><td>Antibody</td></tr><tr><td>CD150 APC clone TC15-12F12.2</td><td>Biolegend</td><td>115909</td><td>Antibody</td></tr><tr><td>CD34 FITC clone RAM34</td><td>BD Bioscience</td><td>553733</td><td>Antibody</td></tr><tr><td>CD45.2 APC clone 104</td><td>Biolegend</td><td>1098313</td><td>Antibody</td></tr><tr><td>MitoSOX Red</td><td>ThermoFisher Scientific</td><td>M36008</td><td>Dye</td></tr><tr><td>Mitotracker Green</td><td>ThermoFisher Scientific</td><td>M7514</td><td>Dye</td></tr><tr><td>Live/dead Yellow Dye</td><td>ThermoFisher Scientific</td><td>L34967</td><td>Dye</td></tr></tbody></table>

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.

Presented is a method for analyzing ROS in the mitochondria of multiple healthy and MLL-AF9-expressing leukemia progenitor populations. Figure 1 displays a schematic view of the protocol workflow, which consists of 4 major steps: 1) BM isolation from mice; 2) Staining BM cells with a fluorogenic dye that recognizes mitochondrial ROS, particularly superoxides; 3) Surface marker antibody staining to delineate various healthy and leukemia hematopoietic populations; and 4) Flow cytometry acquisi...

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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 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.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

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7.3K Views.  Fox Chase Cancer Center. Reactieve zuurstofsoorten of ROS, zijn zeer reactieve zuurstofsoorten die worden geproduceerd door cellen, en beïnvloedt hun gedrag. Hoewel overtollige ROS kan ook leiden tot wijdverbreide cellulaire ravage, en in ernstige gevallen, celdood. Zo is het behoud van ROS homeostase van fundamenteel belang voor cellulaire functie en overleving.Het protocol dat hier wordt gepresenteerd, richt zich op het meten van een specifiek type ROS, dat wordt gegenereerd door mitochondriën, voornamelij...

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

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.

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 ...

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

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

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, 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. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

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.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Developmental Biology

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

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

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

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.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

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

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

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

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

Biology

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Neuroscience

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

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

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.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

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

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