Figure 1.

Isolation of brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes

(A) Overall protocol schematic.

(B) Images of indicated organs without PBS perfusion (right) and with PBS perfusion (left). Scale bar: 2mm.

(C) Photograph of dissociated brain at the end of enzymatic dissociation (note there are no tissue chunks present).

(D) Photographs of Percoll layering sequences (1-4) and layered Percoll gradient before (lower left) and after (lower middle) centrifugation, with enlarged image pointing out the buffy leukocyte layer post-centrifugation (lower right).

(E) Light microscope image of washed leukocytes post-Percoll gradient centrifugation on a hemocytometer (note the lack of debris and single cell nature of suspension). Scale bar: 100mm.

(F) Bar chart showing the percent of viable cells following brain dissociation, leukocyte enrichment, and cell staining, as determined by the percent of cisplatin negative/low cells of all single cells in CyTOF experiments.

See also Methods video S1.

(G) Bar chart derived from CITE-seq data showing percentage of cells sequenced that were leukocytes (CD45+) from the naive and brain metastasis-burdened brain. Error bars represent SEM.

(H) Stacked bar chart derived from CITE-seq data showing proportions of all identifiable cells sequenced that were leukocytes (CD45+), endothelial cells, pericytes, or metastatic cancer cells from the naive and brain metastasis-burdened brain.