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

Reactive oxygen species or ROS, are highly reactive oxygen species that are produced by cells, and influences their behavior. Though excess ROS can also cause widespread cellular havoc, and in severe cases, cell death. Thus, the maintenance of ROS homeostasis is of fundamental importance to cellular function and survival.

The protocol presented here, focuses on measuring a specific type of ROS, that is generated by mitochondria, mainly as a metabolic by-product in live, normal, or healthy hematopoietic stem and progenitor cell populations, as well as in leukemia cells from the mouse model of blood cancer, Acute Myeloid Leukemia or AML. This protocol can also be used to evaluate how genetic manipulation, such as gene deletion or overexpression, impacts mitochondrial ROS in several healthy and malignant hematopoietic populations. And as a result, potentially provide insightful information about redux state and possibly the metabolism of the cells.

This technique authorizes flow settle metric analysis of a protogenic probe to monitor mitochondrial ROS production in live healthy and malignant hematopoietic stem and progenitor sub-populations. This protocol is straightforward and can be completed in a few hours. Moreover, it is suitable for live cell analysis and allows for distinguishing and analyzing mitochondrial ROS in stem cell population in the bone marrow, using surface mark of staining.

After collecting mononuclear bone marrow cells from wild tight mice and leukemic MLL-AF9 mice, according to the manuscript, aliquot 200 microliters of the cell suspension into each of nine single color control tubes. Aliquot the remaining cells into other experimental tubes, 200 microliters each. Then place into a centrifuge two experimental tubes containing bone marrow cells and leukemic cells respectively, and one control tube.

Centrifuge at 300 times G for five minutes. Next, decant the supernatant, and re-suspend the cells in room temperature F-PBS with a live/dead cell stain, according to the manufacturer's instructions. Incubate on ice for 30 minutes.

Afterwards, add 1 milliliter of room temperature F-PBS to the three live/dead stained tubes, and one single color control tube for mitochondrial ROS dye staining. Place the tubes into the centrifuge at 300 times G for five minutes at room temperature. Then, obtain a 5 millimolar mitochondrial ROS dye stock solution by re-suspending 50 micrograms of mitochondrial ROS dye in 13 microliters of DMSO.

Then add 13 milliliters of room temperature F-PBS to the 13 microliter mitochondrial ROS dye to dilute to a final concentration of five micromolar. If necessary, add 13 microliters of 50 millimolar Verapamil to the solution to inhibit mitochondrial efflux pumps. Obtain the tubes from the centrifuge and aspirate off the wash of the live/dead cell stain.

Add 200 microliters of F-PBS in the live/dead staining control and keep it in ice until ready to start the analysis. Add 200 microliters of the mitochondrial ROS dye stain containing Verapamil, to each experimental tube, as well as the mitochondrial ROS staining control tube. Vortex to mix and incubate for 10 minutes at 37 degrees Celsius in the dark.

Then add 1 milliliter of room temperature F-PBS to the control and experimental tubes. Centrifuge five minutes at 300 times G at room temperature. Aspirate off the supernatent and wash the cells with an additional one milliliter of room temperature F-PBS.

Centrifuge again for five minutes at 300 times G, at room temperature. First, prepare two antibody cocktails for healthy and leukemia bone marrow cells. Aspirate the supernatant from the experimental tube containing healthy bone marrow cells, and add 200 microliters of the antibody cocktail number one to the tube.

Vortex to mix. Also prepare the single color control tubes by adding 200 microliters of F-PBS and one microliter of the corresponding antibody. Incubate for 60 minutes on ice in the dark.

Aspirate the supernatant from the experimental tube containing leukemia bone marrow, and add 200 microliters of the antibody cocktail number two, to the tube. Vortex to mix. Incubate for 60 minutes on ice in the dark.

After that, wash all the tubes with 1 milliliter of cold F-PBS and centrifuge for five minutes at 300 times G at room temperature. Re-suspend the cells in 500 microliters of cold F-PBS. Transfer the cell suspension into each flow cytometer tube with a 40 micrometer filter for excluding aggregates.

First, insert one no stain control tube into the flow cytometry machine and start the acquisition to compensate the flow cytometry machine. Repeat for other control tubes. Afterwards, read the experimental tubes to set up the gates.

To analyze the size and complexity of the cell population, first for healthy HSPC, set the forward scatter area and side scatter area plots populations. Press the Square Gate button, to gate out extraneous debris from the forward and side scatter plot. Then, on a forward scatter area and height plot, use a double discriminator to gate out doublets.

Select live cells, lineage low cells, and the various healthy HSPC. For each population of interest, analyze the median fluorescence intensity of the TRPE channel in a histagram plot, to evaluate differences in the mitochondrial ROS signal. Repeat the same procedure of size analysis, gating and histogram plotting for leukemia populations.

Bone marrow cells isolated from healthy mice were stained with a live/dead dye and a mitochondrial ROS dye, and subsequently stained with antibodies recognizing lineage markers:plus CD48, C-kit, Sca-1, CD34 and CD150. In addition, bone marrow cells from leukemia mice were also stained with CD45.2 to discriminate between MLL-AF9 leukemia cells from healthy recipient bone marrow cells stained with CD45.1. A comparison of mitochondrial ROS staining, between healthy CD48 negative LSK, and myeloid progenitors, shows that myeloid progenitors display significantly higher levels of mitochondrial ROS staining.

Moreover, c-Kit high leukemia progenitors display significantly higher levels of mitochondrial ROS staining, compared to c-Kit intermediate low leukemia cells, healthy CD48 negative LSK and myeloid progenitors. C-Kit intermediate low leukemia cells, also displayed a significantly higher value compared to CD48 negative LSK cells, but not to myeloid progenitors. Mitochondrial ROS dyes are highly reactive and appropriate wash steps are needed to ensure that any excess of the dye has been removed before starting the following steps.

There are several chemically distinct ROS fluorogenic dyes that can be used in this protocol, to achieve a bit of knowledge of the redux status in live hematopoietic cells. This technique has been extensively used in the literature, and may provide useful insights on the metabolic and signaling pathways that are differentially regulated in leukemia cells, if compared with their normal counterparts.