This protocol is a simple flow cytometry-based assay allowing measurement of mitochondrial membrane potential and mitochondrial mass in living cells. Standard metabolic assays fail when working with rare populations such as the hematopoietic stem cells. This method enables users mitochondrial membrane profiling when working with low number of cells and to determine novel metabolic modulators of cells such as the HSCs.
Moreover, you can combine it with downstream functional assays such as bone marrow transplantation or colony-forming assays to determine the functionality of your cells post-culture. Our technique can be exploited for the identification of novel molecules capable to improve HSC function in the context of bone marrow transplantation and hematopoietic immune recovery after ablative therapies. As described in our protocol, our method can be used to evaluate metabolic fitness of immune T cells.
It is very important that the cells have minimal exposure to light after staining. Moreover, the use of pump inhibitor verapamil has to be included in order to evaluate the contribution of pump efflux. However, users have to be aware that verapamil profoundly perturbs ionic equilibrium and cannot be used in order to evaluate the effect of a metabolic modulator on mitochondrial membrane potential.
Visual demonstration of this method is extremely important because there are some critical steps that one needs to keep in mind when working with low number of cells, and these steps are very often missing in the regular papers that users read on PubMed. For FAC sorting, the LKS population, defined by lineage negative, Sca1 positive, cKit positive is identified. Hematopoietic stem cells from around five to 10%of the cells in the LKS population are identified by gating for CD150 positive, CD48 negative population, labeled as HSCs.
After hematopoietic stem cell sorting, Centrifuge the tubes at 300 times g for five minutes at four degrees Celsius. Gently remove most of the supernatant without dislodging the pellet, and leave 50 to 80 microliters on top of the cell pellet. Resuspend the cell pellet in stem cell expansion medium to a final volume of 200 microliters.
Prepare a sterile tissue culture treated 96 U-bottom well plate, and identify the wells where the cells will be cultured. Transfer 100 microliters of 2x basal medium previously prepared in these wells. In the NR marked well, add two microliters of a 100x nicotinamide riboside solution.
Transfer 100 microliters of 2x basal medium into wells for staining control. Seed 100 microliters of prepared hematopoietic stem cells on top of the wells containing 2x basal medium. Seed 100 microliters of whole bone marrow cells on wells marked as staining controls.
Add 200 microliters of autoclaved water in all surrounding wells to reduce evaporation from wells containing cells. Place the plate undisturbed in an incubator at 37 degrees Celsius in 5%carbon dioxide for 72 hours. Take out the plate to replenish nicotinamide riboside every 24 hours, and put it back into the incubator.
At the end of the culture period, add two microliters of 100x TMRM solution and two microliters of 100x green fluorescent stain solution in each of the test wells. Add two microliters of 100x TMRM in the TMRM staining control well. Add two microliters of 100x green fluorescent stain in the MitoTracker staining control well.
Cover the top of the plate with aluminum foil. and place the plate back in an incubator at 37 degrees Celsius in 5%carbon dioxide for 45 minutes. Remove the plate from the incubator, and centrifuge it at 300 times g for five minutes.
Invert the plate to remove the supernatant, and add 200 microliters of standard FACS buffer to resuspend. Cover the plate with foil. Centrifuge the plate at 300 times g for five minutes.
Repeat this washing step three times. Resuspend the cells in 200 microliters of FACS buffer, and transfer to FACS tubes. To run the samples on the flow cytometer, first load the single color controls into the machine, and record at least 5, 000 events in each one by one.
In the software, press Acquire and then Record on the dashboard to acquire and record single color controls. Press Compensation Setup, and click Calculate Compensation to calculate the compensation. Once the compensation has been applied, acquire the hematopoietic stem cell sample, and record as many events as possible.
Export the FACS files from the cytometer, and open the file on the analysis software. On FlowJo, use forward scatter and side scatter gating to identify the cell population. Identify singlets in the next gates, and then plot the DAPI negative fraction representing live cells.
In the live cell gate, make a contour plot in the TMRM and green fluorescent stain channel to measure mitochondrial membrane potential and mass, respectively. Export the mean fluorescence intensity of these two channels in the live cell gate. Then, export the proportion of cells in the TMRM low gate in all samples.
The nicotinamide riboside treatment showed a clear increase in the TMRM low population. It also significantly increased the proportion of cells in the TMRM low gate and showed a significant lowering of TMRM fluorescence intensity. Mitochondrial mass did not change upon nicotinamide riboside supplementation.
Additionally, stem cell marker staining was combined with mitochondrial staining post-culture. With the gating strategy to identify hematopoietic stem cells, exposure to nicotinamide riboside showed a significant increase in the TMRM low population and a significant decrease in the TMRM fluorescence intensity in gated HSCs. The green fluorescent mitochondrial stain green signal remained unchanged in the two conditions.
It is important to cover the staining plate with aluminum foil in order to minimize the exposure of light to stained samples. This method can be combined with downstream assays such as bone marrow transplantation and colony-forming assays to determine the functionality of your stem cells. So this technique allows a simple screening method for the identification of novel metabolic modulators of HSCs.
