Open AccessCombined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue
Author(s) -
Madison Trujillo,
Taylor McElroy,
Taurean Brown,
Pilar Simmons,
Fabio Ntagwabira,
Antiño R. Allen
Publication year - 2021
Publication title -
journal of visualized experiments
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.596
H-Index - 91
ISSN - 1940-087X
DOI - 10.3791/63007
Subject(s) - dissociation (chemistry) , cell , pipette , chemistry , biophysics , hippocampal formation , computer science , biological system , neuroscience , biochemistry , biology
This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (>90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.