Our protocol provides the scientist with a powerful tool for marrying multiple micro parameters at single cell level from human IPS and their neuro and clear derivatives. This protocols allow the merriment of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry. This technique can be applied to several cell types including those from other neurodegenerative diseases.
So, it can thereby help provide insight into the mechanisms behind these disease types and also potentially aid in therapeutic screening. To begin, seed the cells separately into four wells in a six well plate and incubate the cells until 50 to 60%confluency is achieved. At the end of the culture period, prepare five individual staining solutions containing different combinations of culture medium, FCCP, TMRE, MTG, and mito sock spread.
Add them into the respective wells, and incubate the cells as described in the manuscript. Next, aspirate the medium from all the wells and wash with PBS. For cell detachment, incubate the cells with one milliliter of cell dissociation reagent at 37 degree Celsius for five minutes.
Neutralize the cell dissociation reagent with one milliliter of DMEM, supplemented with 10%FBS. Then collect the well content in a 15 milliliter chronicle tube. Pellet down the cells by centrification and wash the cell pellet with PBS once or twice.
Aspirate all the supernatant, leaving approximately 100 microliters in the tube. Re-suspend the cell pellets in 300 microliters of PBS. After transferring the cell suspension into a 1.5 milliliter micro centrifuge tube, keep the tube in the dark at room temperature.
Analyze the cells using the flow cytometer with the band pass filter settings described in the text manuscript. Detach approximately 1 million cells using the cell dissociation reagent and pellet down the cells as demonstrated previously. Neutralize the cell suspension with DMEM plus 10%FBS and collect the suspension in a 15 milliliter tube.
Fix the cells with one milliliter of 1.6%paraformaldehyde at room temperature for 10 minutes. Permeabolize the cells with one milliliter of ice cold 90%methanol at minus 20 degrees Celsius for 20 minutes. Then block the samples in one milliliter of blocking buffer, and wash the cells by centrifugation with PBS, as demonstrated.
To detect different mitochondrial complexes and subunits, incubate the cells for 30 minutes with the respective primary antibodies described in the text manuscript. After washing the cells once with PBS, incubate the cells with secondary antibody for 30 minutes. At the end of the incubation, wash and re-suspend the cells and PBS as demonstrated.
Transfer the cell suspension into a 1.5 milliliter micro centrifuge tube, kept in the dark on ice. Next, analyze the cells on the flow cytometer and detect signals and filter one using a 5 30/30 band pass filter. For each cell subpopulation, select a histogram plot and analyze the median fluorescence intensity or MFI of the different filter channels.
Calculate the TMRE levels, specific values for MMP ROS complex subunit and TFAM, as described in the text manuscript. Flow cytometry and live cells was used to investigate MMP mitochondrial volume and ROS levels. In POLG DA neurons, lower specific MMP and higher specific ROS was observed, while no changes in mitochondrial volume, total MMP and ROS.
POLG astrocytes had decreased MMP, but no change in mitochondrial volume and ROS. Flow cytometry and fixed cells was used to investigate the level of MRC complex subunit and TFAM. DA neurons showed increased complex one and TFAM versus control, but no change in complex two level.
POLG astrocytes showed a reduction of complex one four and specific TFAM. As the cell density can also influence MMP and the relationship between MTG and MMP fluorescence, this is cell specific. Adapting this protocol to other cell types will likely require optimization Following this procedure, Microscopic based asis, for example confocal microcopy can be performed, providing additional details of mitochondrial structures.
So, while this strategy detects mitochondrial dysfunction in DA neurons and astrocytes from a known mitochondrial disease, these techniques can also be applied to explore mitochondrial function in any type of cell and disease.
