To begin, take the skeletal muscle cells stained with cisplatin, metal conjugated antibodies, and intercalator-iridium solution, and pellet the cells by centrifugation. Once the supernatant is removed, add one milliliter of CSM to the vortexed cells. Pellet the cells by centrifugation and wash in one milliliter of CAS buffer.
After centrifugation, aspirate the supernatant to approximately 200 microliters. Add one milliliter of CAS buffer to the vortexed samples and aliquot five microliters for cell count. After centrifuging the cells and aspirating the supernatant to 50 microliters, resuspend the cells in CAS buffer to a final concentration of 1 million cells per milliliter and add calibration beads to achieve a final concentration of 0.1x.
Load the sample into the mass cytometer to collect data using a flow rate of 400 to 500 cells per second, and normalize the data after collection. If necessary, concatenate individual Flow Cytometry Standard or FCS files for each sample into a single file. Identify single cells by gating on iridium intercalator positive events.
Select events that are negative for cisplatin to gate for live cells. Gate on the population of interest and quantify the relative proportion of stem and progenitor cells. To perform high-dimensional analysis, export the population of interest and use clustering algorithms.
For X-shift analysis, download the Vortex software package and Java 64-bit. Upload the exported cell populations to a local database and define the clustering parameters. To visualize the spatial relationships between cell populations within the X-shift clusters, perform a force-directed layout.
CyTOF analysis identified a sequence of four populations, stem cells, and progenitor populations one through three. The progenitor populations P1 and P2 correspond to increasingly more mature progenitor cells. P3 was previously identified but not characterized due to its low abundance.
The stem and progenitor cell dynamics post-injury were analyzed using CD9 by CD104 by axial dot plots. A notable increase in the stem cell population was observed on day three, suggesting expansion. This was supported by increased iododeoxyuridine incorporation, indicating cell proliferation, and heightened MyoD expression as shown by the color overlay.
Activated stem cells marked by high levels of CD98, CD44, MyoD, and iododeoxyuridine were identified, underscoring their high proliferation and activated state at day three post-injury.
