Microglia Isolation and Multiparameter Staining

The microglia were dissociated with trypsin and collected with completed DMEM. Cells were centrifuged at 1500 rpm and resuspended in FACS buffer before being moved to 5 ml of FACS tubes for staining. Microglia cells were incubated in blocking buffer (CD16/32-APC; 1:100; BioLegend) on ice for 10 min to block Fc receptor. Surface antibody cocktails were prepared with CD45/PerCP-Cy5.5 (1:100; BioLegend) and CD11b/APC-Fire 750 (1:100; BioLegend) including viability dye Zombie Aqua. Intracellular staining was performed with MAP2/Alexa Fluor 594 (1:100) and myelin CNPase/Alexa Fluor 647 (1:100).

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Ritzel R.M., Li Y., Jiao Y., Lei Z., Doran S.J., He J., Shahror R.A., Henry R.J., Khan R., Tan C., Liu S., Stoica B.A., Faden A.I., Szeto G., Loane D.J, & Wu J. (2023). Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration. Science Advances, 9(10), eadd1101.

Publication 2023

CD45/PerCP-Cy5.5 CD11b/APC-Fire 750

Alexa 594 Alexa fluor 647 Antibody cocktails Block Buffer Cd11b Cells Cnpase Cy5 5 Fc receptor Intracellular Map2 Microglia cells Myelin Trypsin

Corresponding Organization : Trinity College Dublin

Other organizations : The University of Texas Health Science Center at Houston, Howard University, Allen Institute for Immunology, University of Washington

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Variable analysis

independent variables

Trypsin dissociation of microglia
Staining with surface antibody cocktails (CD45/PerCP-Cy5.5, CD11b/APC-Fire 750, and viability dye Zombie Aqua)
Intracellular staining with MAP2/Alexa Fluor 594 and myelin CNPase/Alexa Fluor 647

dependent variables

Microglial cell characteristics after dissociation and staining

control variables

Centrifugation at 1500 rpm
Resuspension in FACS buffer
Incubation in blocking buffer (CD16/32-APC; 1:100; BioLegend) for 10 min to block Fc receptor

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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