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-regu....

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

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