Cell Surface Marker Analysis by Flow Cytometry

For cell surface flow cytometry, cells were incubated with human Fc receptor binding inhibitor (#14-9161−73; eBioscience) followed by primary antibodies CD235−APC (#306607; BioLegend) CD41-FITC (#303703; BioLegend) and CD11b-PE/Cy5 (#101209; BioLegend) or CD-71-APC (BD551374; BD Biosciences), p53-PECy7 (NB200-171; Novus), Sca-1-PE (#12-5981-81; eBioscience), CD34-Fluor®450 (#48-0341-82; eBioscience) and CD16/32-FITC (#101305; BioLegend). FRET measurements were obtained as described^{69 (link)}. To measure ECFP and FRET cells were excited with the 405 nm laser and fluorescence was collected in the ECFP channel with a standard 450/40 filter, while the FRET-signal was measured with a 529/24 filter. To measure EYFP, cells were excited with the 488 nm laser while emission was also taken with a 529/24 filter. Data were collected on a DxP10 flow cytometer (Cytek) and analyzed by using FlowJo Software, v.9.7.2.

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Wilkes M.C., Siva K., Chen J., Varetti G., Youn M.Y., Chae H., Ek F., Olsson R., Lundbäck T., Dever D.P., Nishimura T., Narla A., Glader B., Nakauchi H., Porteus M.H., Repellin C.E., Gazda H.T., Lin S., Serrano M., Flygare J, & Sakamoto K.M. (2020). Diamond Blackfan anemia is mediated by hyperactive Nemo-like kinase. Nature Communications, 11, 3344.

Publication 2020

human Fc receptor binding inhibitor CD235−APC CD41-FITC CD11b-PE/Cy5 CD-71-APC Sca-1-PE CD34-Fluor®450 CD16/32-FITC DxP10 flow cytometer

Antibodies Cd11b Cells Fc receptor Fitc Flow cytometry Fluorescence Fret Human Novus Sca 1

Corresponding Organization :

Other organizations : Stanford University, Lund University, Institute for Research in Biomedicine, Science for Life Laboratory, Karolinska Institutet, SRI International, Broad Institute, University of California, Los Angeles, Institució Catalana de Recerca i Estudis Avançats

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Variable analysis

independent variables

Primary antibodies CD235-APC, CD41-FITC, CD11b-PE/Cy5 or CD-71-APC, p53-PECy7, Sca-1-PE, CD34-Fluor®450, and CD16/32-FITC

dependent variables

FRET measurements

control variables

Human Fc receptor binding inhibitor (#14-9161-73; eBioscience)
Cells were excited with the 405 nm laser and 488 nm laser
Fluorescence was collected in the ECFP channel with a standard 450/40 filter, while the FRET-signal was measured with a 529/24 filter
EYFP emission was also taken with a 529/24 filter
Data were collected on a DxP10 flow cytometer (Cytek) and analyzed by using FlowJo Software, v.9.7.2

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