Analysis of Immune Cells in Mouse Nervous Tissues Using Flow Cytometry

Take cells isolated from mouse sciatic nerves and dorsal root ganglia or DRGs.               

Incubate with fluorophore-tagged antibodies against CD45 and CD11b, and with biotin-tagged antibodies against MHC class II, to label immune cells.

Incubate with fluorophore-tagged streptavidin that binds to biotin.

Treat with a fixative to stabilize the cellular structure.

Introduce a permeabilization buffer with a fluorescent DNA stain to permeabilize membranes and label the nuclei.

Using a flow cytometer, pass the cells through the laser beam to assess optical and fluorescence characteristics.

Remove aggregates with a higher DNA fluorescence to isolate single cells and detect the fluorescence from the markers.

Identify cells expressing CD45, a characteristic marker of immune cells, confirming their presence.

Most CD45-positive cells in the DRG express CD11b and MHC class II, indicating microglia-like cells, which are absent in the sciatic nerves.

This technique complements imaging for analyzing immune cell heterogeneity within nervous tissues.

At the computer, select the appropriate channels for detection of the fluorophores of interest, the appropriate flow rate for the samples, and the total number of events to be collected per sample. The minimum number of events should be 1 million.

Next, create scatter plots under a global worksheet of the side scatter A versus the forward scatter A, as well as for each fluorophore of interest versus the forward scatter A. Also, create a statistical view that will display the number of events and the mean fluorescence intensity for the selected fluorophores.

Determine the parent gate P1 based on the scatter plot from DAPI-only staining controls. Attach the sample to flow cytometry and click on Acquire. Observe the DAPI versus FSC plot, and reduce the voltage of the DNA Pacific Blue channel until the DAPI-plus population becomes visible. Using the rectangle gating tool, draw a box around the DAPI-plus population. This represents the P1 gate. Select the scatter plots for each of the fluorophores and display only the DAPI-plus events. This represents the background fluorescence for each of the fluorophores.

Using the rectangle gating tool, draw a box in the scatter plots for each of the fluorophores, starting at 103 on the y-axis. This gate represents the region in which positive events for each of the stated markers should be found. Adjust the voltages for each of the fluorophores using the DAPI-only staining controls. Attach the sample to flow cytometry and click on Acquire.

Observe the position of the DAPI-plus population for each of the fluorophores, and adjust the respective voltages such that the populations fall outside of the rectangle gate. Once the flow cytometer has been set up, run the samples and export the forward scatter files for further analysis.