Multicolor Flow Cytometry Analysis

Blood was collected into EDTA tubes via cardiac bleeds and prepared using Dako Uti-Lyse erythrocyte lysing solution according to instructions. Blood was stained with AF647-labeled CSF1-Fc (6 (link)) and the following Abs: CD32^APC (Clone REA256, 1:40) and CD161^APC (Clone REA227, 1:40) from Miltenyi Biotec, B220^PE (Clone His24, 1:400; eBioscience), CD4^FITC (Clone W3/25, 1:10; Bio-Rad Laboratories), CD3^AF488 (Clone 1F4, 1:200), and SIRPα^PE (Clone OX-41, 1:400) from BioLegend. Myelin-depleted single-cell suspensions of brain were prepared as described in (21 (link)), except rats were perfused with physiological saline and heparin and then stained with CD45^EF450 (Clone OX1, 1:200; eBioscience) and CD11b/c^AF647 (Clone OX-42, 1:500; BioLegend). Peritoneal cavity cells were isolated as described in (22 (link)) and stained with SIRPα^PE and CD11b/c^AF488 as described above. Isotype controls were used for all flow cytometry data. Cells were analyzed by flow cytometry on a FACSCalibur, LSRFortessa X-20 or LSRFortessa (BD Biosciences). Analysis was performed with FlowJo software (FlowJo).

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Pridans C., Raper A., Davis G.M., Alves J., Sauter K.A., Lefevre L., Regan T., Meek S., Sutherland L., Thomson A.J., Clohisey S., Bush S.J., Rojo R., Lisowski Z.M., Wallace R., Grabert K., Upton K.R., Tsai Y.T., Brown D., Smith L.B., Summers K.M., Mabbott N.A., Piccardo P., Cheeseman M.T., Burdon T, & Hume D.A. (2018). Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus. The Journal of Immunology Author Choice, 201(9), 2683-2699.

Publication 2018

Uti-Lyse erythrocyte lysing solution CD161APC B220PE CD4FITC CD3AF488 SIRPαPE CD45EF450 CD11b/cAF647 FACSCalibur LSRFortessa X-20 LSRFortessa

Af647 Bleeds Blood Brain Cardiac Cd11b Cells Clone Csf1 Edta Erythrocyte Flow cytometry Heparin Isotype Myelin Peritoneal cavity Physiological Rats Saline

Corresponding Organization : Mater Research

Other organizations : New World Laboratories, John Radcliffe Hospital, University of Oxford, MRC Centre for Reproductive Health, Medical Research Council, University of Newcastle Australia

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Variable analysis

independent variables

Staining with AF647-labeled CSF1-Fc
Staining with the following Abs: CD32^APC, CD161^APC, B220^PE, CD4^FITC, CD3^AF488, SIRPα^PE, CD45^EF450, CD11b/c^AF647, CD11b/c^AF488

dependent variables

Cell population analysis by flow cytometry

control variables

Blood collected into EDTA tubes via cardiac bleeds
Blood prepared using Dako Uti-Lyse erythrocyte lysing solution
Myelin-depleted single-cell suspensions of brain prepared as described in (21)
Peritoneal cavity cells isolated as described in (22)
Isotype controls used for all flow cytometry data
Cells analyzed by flow cytometry on a FACSCalibur, LSRFortessa X-20 or LSRFortessa (BD Biosciences)
Analysis performed with FlowJo software (FlowJo)

controls

Isotype controls used for all flow cytometry data

Annotations

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