This protocol may be useful for addressing questions about the role of the choroid plexuses and peripheral immune cells in regulation of the brain in various contexts in mouse models. This protocol allows for in-depth quantitative and qualitative analysis of the unique immune cell populations in the choroid plexuses of mice. Dissection of the choroid plexuses can be difficult at the beginning due to their small size and transparent color.
We recommend practicing on non-perfused animals first. To begin, position the brain dissected from a C57 black six mouse dorsal side up in a Petri dish and place the dish below the objectives of the binocular loops. While maintaining the brain in place using forceps, insert the two ends of another pair of forceps down through the midline between the hemispheres.
Using the forceps, pull the cortex with the callosum and hippocampus away from the septum, exposing the lateral ventricle and a part of the third ventricle. Identify the lateral choroid plexus as a long veil lining the lateral ventricle and flaring at both ends. Then using two ends of thin forceps, collect the lateral choroid plexus.
Be careful to collect the triangular posterior part hidden by the posterior fold of the hippocampus. Next, pull the cortex with the corpus callosum and hippocampus of the contralateral hemisphere away from the septum to expose the entire third ventricle and the opposite lateral ventricle. Using fine forceps, collect the third choroid plexus identified as a short structure with a granular surface aspect.
Then collect the other lateral choroid plexus. Next, insert the two ends of the forceps down between the cerebellum and midbrain and detach the cerebellum from the pons and medulla to expose the fourth ventricle. Then identify the fourth choroid plexus as a long globular structure with a granular surface aspect that lines the fourth ventricle from the lateral right end to the left end between the cerebellum and medulla.
Using fine forceps, collect the fourth choroid plexus. After incubating all collected choroid plexuses with collagenase IV, gently pipette up and down around 10 times to finalize the dissociation. Then fill the tube to 1.5 milliliters with MACS buffer to stop the collagenase IV activity.
Next, centrifuge the cells and discard the supernatant before washing the cells with 1.5 milliliters of MACS buffer. After adding the appropriate compensation beads and antibody solutions, incubate the samples on ice for 30 to 45 minutes, protecting them from light exposure. Then fill the tubes with 1.5 milliliters of MACS buffer and centrifuge the cells and compensation beads.
After resuspending the cells and compensation beads in 500 microliters of MACS buffer, filter the cells through a 70 micron strainer. After turning on the flow cytometer and the software, select forward scatter area versus side scatter area and gate for cells based on size. Then create a daughter gate for single cells with forward scatter area versus forward scatter height.
Next, create a daughter gate for live cells with DAPI versus forward scatter area to exclude the DAPI positive cells. Then create a daughter gate for CD45 positive immune cells with CD45 versus forward scatter area. Now, create a daughter gate from the CD45 positive cells and select F480 versus CD11b to identify the CD11b positive F480 positive and the CD11b positive F480 negative populations.
Then create a daughter gate for CD11b positive F480 positive cells and select CX3CR1 versus IA-IE to identify both CX3CR1 positive IA-IE positive border-associated macrophages and CX3CR1 positive IA-IE negative macrophages. Next, create a daughter gate for CD11b positive F480 positive CX3CR1 positive IA-IE negative macrophages and select F480 versus CD11b to identify both the CD11b high F480 intermediate CX3CR1 positive IA-IE negative Kolmer's epiplexus macrophages and the CD11b positive F480 high CX3CR1 positive IA-IE negative border-associated macrophages. Then from the CD11b positive F480 negative population, gate for Ly6C versus IA-IE.
Select both IA-IE positive Ly6C negative population and IA-IE negative Ly6C positive cells that mainly correspond to monocytes or neutrophils. Finally, create a daughter gate for CD11b positive F480 negative IA-IE positive Ly6C negative population and select CD11b versus CX3CR1. Then identify both CD11b high CX3CR1 low and CD11b positive CX3CR1 negative populations.
For T-cell gating, after excluding the DAPI positive cells as described earlier, create a daughter gate for CD45 positive immune cells with CD45 versus FSC-A, then create a daughter gate from the CD45 positive cells and select TCR beta versus CD11b. Exclude the CD11b positive myeloid cells and select the TCR positive CD11b negative T-cell population. Finally, create a daughter gate for the TCR beta positive CD11b negative population and select CD4 versus CD8a.
Identify both CD8 negative and CD4 positive T-cells and CD8 positive CD4 negative T-cells. The flow cytometry analyses demonstrated here successfully revealed major subsets of myeloid and T-cells and their relative total number per mouse in a highly reproducible manner. Flow cytometry analysis of the myeloid cells show that the choroid plexus is populated by CD11b positive CX3CR1 positive F480 high border-associated macrophages, representing 80%of the CD45 positive immune cells at the choroid plexus.
The CD11b high F480 intermediate CX3CR1 positive IA-IE negative population of Kolmer's epiplexus macrophages was also identified. Among the CD11b positive F480 negative immune cells, two different populations of Ly6C negative IA-IE positive cells were characterized and likely correspond to dendritic cells. The analysis and gating strategy of T-cells highlighted the presence of the minor population of CD45 positive CD11b negative TCR beta positive cells.
Among them, about 30-50 CD8 negative CD4 positive and CD8 positive CD4 negative T-cells per mouse populated the choroid plexus under physiological conditions. It is important to control for the quality of the perfusion since any blood contamination may blur the immune composition of the choroid plexus. This method can be followed by sorting of selected cell populations for further analysis such as bulk or single-cell RNA sequencing.
