Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses
Efthalia Preka,
Alejandro Lastra Romero,
Ying Sun,
Yara Onetti Vilalta,
Thea Seitz,
Adamantia Fragkopoulou,
Christer Betsholtz,
Ahmed M. Osman,
Klas Blomgren
Affiliations
Efthalia Preka
Department of Women's and Children's Health, Biomedicum A4, Karolinska Institutet, 171 77, Stockholm, Sweden
Alejandro Lastra Romero
Department of Women's and Children's Health, Biomedicum A4, Karolinska Institutet, 171 77, Stockholm, Sweden
Ying Sun
Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Sweden
Yara Onetti Vilalta
Department of Women's and Children's Health, Biomedicum A4, Karolinska Institutet, 171 77, Stockholm, Sweden
Thea Seitz
Department of Women's and Children's Health, Biomedicum A4, Karolinska Institutet, 171 77, Stockholm, Sweden
Adamantia Fragkopoulou
Department of Women's and Children's Health, Biomedicum A4, Karolinska Institutet, 171 77, Stockholm, Sweden
Christer Betsholtz
Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Sweden; Department of Medicine Huddinge, Campus Flemingsberg, Neo, 141 57, Huddinge, Sweden
Ahmed M. Osman
Department of Women's and Children's Health, Biomedicum A4, Karolinska Institutet, 171 77, Stockholm, Sweden
Klas Blomgren
Department of Women's and Children's Health, Biomedicum A4, Karolinska Institutet, 171 77, Stockholm, Sweden; Pediatric Oncology, Karolinska University Hospital, 171 64, Stockholm, Sweden; Corresponding author. Karolinska Institutet, Department of Women's and Children's, Biomedicum A4, 171 65, Stockholm, Sweden.
Cell isolation protocols from brain tissue include prolonged ex vivo processing durations, rendering them suboptimal for transcriptomic studies. Particularly for microglia and vascular cells, current isolation methods produce lower yields, necessitating addition of an enrichment step, and use of large tissue volumes - in most cases whole brain tissue - to obtain sufficient yields. Here, we developed a simple, rapid, and reproducible cell isolation method for generating single-cell suspensions from micro-dissected brain regions, enriched for microglia and vascular cells, without an enrichment step. Cells isolated using this method are suitable for molecular profiling studies using 10 × Genomics Chromium single-cell RNA sequencing with high reproducibility. Our method is valuable for longitudinal unbiased molecular profiling of microglia and vascular cells within different brain regions, spanning multiple time points across physiological development or disease progression.
