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

Summary

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.

Abstract

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.

Introduction

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 tumors^2,3,4. 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 survival^7,8. Importantly, strategies for therapeutically capitalizing on these redox differences have shown promise in several human cancer settings^9,10. 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.

Protocol

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.

1. Recover mono-nuclear bone marrow cells, as described previously^12,13,14,15, 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 crushing^12,13 or by flushing bones^14,15. For the experiments presented here, BM was recovered from both healthy and leukemia mice via flushing.

2. Mitochondrial ROS Fluorogenic Dye Staining

1. 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.
2. 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.
3. Aliquot 200 µL 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: (Optional) A positive control for the induction of mitochondrial ROS can be prepared by treating 2 x 10⁵ cells in 200 µL with 20 µM of Menadione Sodium Bisulfite (MSB) for 1 h at 37 ˚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 µL with 20 µM of MSB plus 100 µM N-acetyl-L-cysteine (NAC) for 1 h at 37 ˚C in a 5% CO₂ incubator.
4. Aliquot the remaining cells in a tube (experimental tube) and centrifuge at 300 x g for 5 min.
5. Resuspend the cells in F-PBS with a live/dead cell stain according to the manufacturer’s instructions. Incubate on ice for 30 min. Be sure to add live/dead stain to the single-color control tube.
6. 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.
7. Resuspend 50 µg of the mitochondrial ROS dye in 13 µL of dimethyl sulfoxide (DMSO) to obtain a 5 mM stock solution.
8. Dilute mitochondrial ROS dye to a final concentration of 5 µM in RT F-PBS with or without Verapamil (50 µM).
9. Aspirate off the wash of the live/dead cell stain. Add 200 µL of mitochondrial ROS dye stain containing Verapamil to each experimental tube as well as the mitochondrial ROS dye single-color control tube.
10. Vortex to mix and incubate for 10 min at 37 ˚C in the dark.
11. 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.
12. 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.

3. Lineage Antibody Staining

1. Prepare the antibody cocktails listed in Table 1.
   NOTE: These antibodies cocktails have been optimized previously^14,15,16.
2. Aspirate the supernatant from the final mitochondrial ROS dye wash of the experimental tubes containing healthy BM and add 200 µL 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.
3. Aspirate the supernatant from the final mitochondrial ROS dye wash of the experimental tubes containing leukemia BM and add 200 µL of the antibody cocktail #2 to each tube. Vortex to mix. Incubate for 60 min on ice in the dark.
4. Wash with 1.0 mL of cold F-PBS and centrifuge 5 min at 300 x g at RT.
5. Resuspend cells in 500 µL of cold F-PBS and filter the cells in a flow cytometer tube using a 40 µm filter to exclude aggregates.

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.

1. 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.
2. Use the no-stain and single-color control tubes to compensate the flow cytometer.
3. Gate out extraneous debris from the forward and side scatter plot (Figure 2A,B, first panel from the left).
4. Gate out doublets using a double discriminator such as the forward discriminator (Figure 2A,B, second panel from the left).
5. Follow the gating strategy proposed in Figure 2A,B to select live cells, lineage low cells and the various HSPC and leukemia subsets.
6. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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