During hematopoiesis, hematopoietic stem cells undergo a metabolic switch from glycolysis to oxidative phosphorylation. This protocol can be used to assess glycolytic and mitochondrial functions of mouse hematopoietic stem and progenitor cells. This method can be used to assess cellular bioenergetics in real time.
The 96-well microplate-based platform offers high throughput quantification with high sensitivity, allowing simultaneous analysis of multiple samples using a single plate. This technique can be used to probe the effects of chemicals or genetic manipulations on HSC metabolism under varied conditions. It can help elucidate metabolic pathways that sustain HSC pluripotency.
Although the current study is mainly focused on mouse hematopoietic stems and progenitor cells, the protocol and this approach could easily be adapted to optimize analysis conditions for any type of suspension cells. One day before the assay, open the extracellular flux assay kit to remove the sensor cartridge and utility plate assembly. Save the loading guide flats for use the next day.
Now manually separate the sensor cartridge from the utility plate and place it upside down next to the utility plate. Using a multichannel pipette, fill each well of the utility plate with 200 microliters of calibrant included with the flux assay kit, then place the sensor cartridge back onto the utility plate, making sure to completely submerge the sensors in the calibrant. Then incubate the utility plate with calibrant and assembled sensor cartridge in a non-carbon dioxide incubator at 37 degrees Celsius overnight following the humidifying conditions mentioned in the manuscript.
On the day of the assay, prepare 2.5 milliliters of the cell adhesive solution per 96-well cell culture microplate as described in the text manuscript. Open the 96-well cell culture microplate included with the flux assay kit and dispense 25 microliters of the prepared cell adhesive solution at the bottom of each well. Cover the microplate with the lid and incubate for 30 minutes at room temperature inside the hood.
After incubation, remove and discard the excess cell adhesive solution using a multichannel pipette or aspirator and wash each well twice with 200 microliters of sterile ultrapure water. Air dry the plate without the lid inside the hood for 30 to 45 minutes. Centrifuge the prepared mouse lineage negative HSPCs at 200 times G for five minutes at room temperature.
After discarding the supernatant, resuspend the cell pellet in the appropriate assay medium, and again centrifuge the cell suspension. Repeat this procedure one more time and resuspend the cells in the appropriate warmed assay medium to the concentration of 250, 000 cells per 50 microliters or five million cells per milliliters. Use a multichannel pipette to add 50 microliters of the cell suspension along the side of each well of the cell adhesive coated 96-well cell culture microplate, then add 50 microliters of the assay medium into the corner background measurement wells.
Create a centrifuge balance plate by adding 50 microliters of water per well of a non-coated 96-well cell culture microplate. Centrifuge the cells at 200 times G for one minute at room temperature. Incubate the plate in a non-carbon dioxide incubator at 37 degrees Celsius for 25 to 30 minutes for cell attachment.
After 30 minutes, visually confirm under a microscope that the cells are stably adhered to the microplate surface. Begin by preparing all the required solutions in the pre-warmed glycolysis stress test assay medium as mentioned in the text manuscript and remove the hydrated sensor cartridge from the incubator. Lift the sensor cartridge out of the calibrant and replace it on the same utility plate with the calibrant to remove the air bubbles.
Place the 80 loading guide flat on the top of the sensor cartridge orienting it so that the letter A is located on the upper left-hand corner. Using a multichannel pipette, dispense 20 microliters of 100 millimolar glucose solution in port A.Replace the 80 loading guide flat with the BC loading guide flat, orienting the letter B on the upper left-hand corner. Dispense 22 microliters of 20 micromolar oligomycin solution in port B.Reorient the BC loading guide flat to locate the letter C on the upper left-hand corner for loading port C and dispense 25 microliters of 500 millimolar 2-deoxy-d-glucose solution in port C.Then remove and discard the loading guide flats.
Now create or load the template for the glycolysis stress test on the controller. Enter the details regarding injection strategies, treatment conditions, and cell types, and press Generate Groups. Go to the plate map and assign wells to each group to be analyzed.
In the protocol, ensure that calibration and equilibration are checked in the initialization step. For the baseline measurement and measurements after each injection, set the number of measurement cycles to three and mix, wait, measure times to three minutes, zero minutes, three minutes. Click Start Run on the software.
Remove the lid from the compounds loaded and hydrated sensor cartridge and place it on the work tray of the extracellular flux analyzer. Begin the calibration run. Take out the microplate containing the seeded cells from the incubator.
Without disturbing the cells, slowly add 130 microliters of the pre-warmed glycolysis stress test assay medium per well to make up the medium volume in each well to 180 microliters and return the plate to the incubator for an additional 15 to 20 minutes. Click Open Tray on the software to open the thermal tray of Seahorse. After the calibration is over, replace the utility plate with this assay microplate containing the cells and press the Load Cell Plate to start the measurements.
After the measurements are over, remove the assay medium from the plate without disturbing the cells and wash them gently with 250 microliters of phosphate buffered saline without dislodging the cells. Now add 10 microliters of radioimmunoprecipitation lysis buffer supplemented with 1X protease inhibitor cocktail to each well and agitate the plate on a shaker for 10 minutes. Freeze the whole plate at minus 80 degrees Celsius.
Thaw the plate and perform a protein measurement assay for data normalization following the manufacturer's instructions. Retrieve and analyze the data by opening the data file using Wave desktop software. Click on Normalize, paste the normalization values for each well and then click on Apply to normalize the data.
Click on Export and select glycolysis stress test report generator to export the analyzed data to a report generator. The non-glycolytic acidification rate rises with increased cell number from 50, 000 to 250, 000, but increases only minimally between 200, 000 to 250, 000. Injection of 10 millimolar glucose stimulates glycolysis at all cell numbers with a maximum increase in ECAR observed at 250, 000 cells per well.
However, injection of two micromolar of oligomycin does not further increase ECAR. The OCR data obtained in the same set of glycolysis stress tests show a significant drop following oligomycin injection due to complex 5 inhibition. Glycolysis stress test parameters were calculated and 250, 000 cells per well in two micromolar of oligomycin were selected for further studies.
The mitochondrial stress test showed that two micromolar oligomycin injection causes a significant reduction in OCR via the inhibition of complex 5. FCCP stimulates OCR in HSPCs in a dose-dependent manner with a maximal increase observed at two micromolar. A mixture of 0.5 micromolar rotenone and 0.5 micromolar antimycin A injection decreases OCR to its minimal level corresponding to non-mitochondrial oxygen consumption.
Mitochondrial stress test parameters were calculated and two micromolar of FCCP was selected for further studies. Given the high throughput nature of this technique, the protocol described here could easily be adapted to screen a large number of bioenergetic modulators in hematopoietic stem cells, hematopoietic progenitors, and malignant hematopoietic cells. Seahorse analysis is widely used in the literature to measure metabolism in a wide variety of contexts in the presence of various substrates and inhibitors.
