All procedures have been described before (23 (link)). All antibody labeling was performed on ice for 20 min, followed by washing and centrifugation. Cells were stained with directly conjugated antibodies to mouse CD45 (30-F11-APC, eBioscience), mouse CD31 (390-APC, BioLegend), Ter119 (TER-119-APC, eBioscience), and human HLA-A, B, C (G46-2.6-BV421, BD Biosciences) to select live human melanoma cells and exclude endothelial and hematopoietic cells. Cells were examined on an LSRFortessa cell analyzer (BD Biosciences) or sorted on a FACSAria cell sorter (Becton Dickinson). SETDB1 or EZH2 knockdown cells were sorted as DsRed and green fluorescent protein (GFP) double-positive cells.
Protocol detail
Human Melanoma Cell Isolation and Sorting
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Antibodies
Antibody
Cells
Centrifugation
Endothelial
Ezh2
Green fluorescent protein
Hematopoietic
Human
Melanoma
Mouse
Setdb1
Corresponding Organization : Children's Medical Center
Other organizations : Howard Hughes Medical Institute
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Variable analysis
independent variables
- SETDB1 or EZH2 knockdown
- Live human melanoma cells selected
- Endothelial and hematopoietic cells excluded
- SETDB1 or EZH2 knockdown cells sorted as DsRed and green fluorescent protein (GFP) double-positive cells
- Antibody labeling procedures
- Staining with directly conjugated antibodies
- Cell examination on an LSRFortessa cell analyzer or sorting on a FACSAria cell sorter
- Labeling with antibodies to mouse CD45, mouse CD31, Ter119, and human HLA-A, B, C
- Not explicitly mentioned
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