Characterization of adherent cultures required dissociation into single cells by incubation with prewarmed TrypLE-select™ at 37 °C. Incubation time varied with the type of cell culture; 5 minutes for neonatal human thymus-derived TEC cultures and 10-15 minutes for and PSC-derived FOXN1+ cultures. Conjugated monoclonal mouse anti-human antibodies: CD104-APC (1:50, clone 422325, Invitrogen), EPCAM-PeCy7 (1:200, clone 12c2, BioLegend), EPCAM-BV421(1:50, clone 9C4, BioLegend), CD90-PE (1:100, clone 5e10, Biolegend), CD90-BV421 (1:50, clone 5e10, Biolegend) were diluted in FACS wash buffer (PBS supplemented with 5% fetal bovine serum) and incubated with cells for 20 minutes on ice. The cell suspension was washed twice with FACS wash solution to remove unbound antibodies and resuspended in FACS wash solution containing 1 μg/ml propidium iodide. Cell surface staining was examined using a Becton Dickenson (BD) LSRFortessa Cell Analyzer. Flow cytometry data were analyzed using the FlowLogic program (7.2.1, DataNova). Alternatively, cell purification was performed using a BD FACSaria FUSION or Influx cell sorter based on cell surface staining or the expression of a fluorescent reporter. Cells were collected using a 5ml FACS tube containing 0.5ml cold fetal calf serum.
Cd104 apc
Manufactured by Thermo Fisher Scientific
CD104-APC is a fluorescently-conjugated antibody that binds to the CD104 cell surface antigen. It is used for the identification and analysis of CD104-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd90 pe, by BioLegend (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
Epcam pe cy7, by BioLegend (1 mentions)
Epcam bv421, by BioLegend (1 mentions)
Cd90 bv421, by BioLegend (1 mentions)
Cd44 pe cy7, by BioLegend (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
2 protocols using cd104 apc
1
Dissociation and Flow Cytometry of Cultured Cells
2
Generating Single-Cell Clones from SUM149PT Cell Line
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The parental cell line SUM149PT was maintained in standard media. To generate single-cell clones, fluorescence-activated cell sorting (FACS) was performed on SUM149PT with the FACSAria III Cell Sorter. Cells were stained with CD44-PeCy7 (1:100; BioLegend, 103030), CD104-APC (1:200; Invitrogen, #50-1049-82), or Epcam-BV510 (1:100; BioLegend, #324235) for 30 min on ice before the addition of 4′,6-diamidino-2-phenylindole (DAPI) (1:1000; Sigma-Aldrich 10236276001; 10 mM stock). Gating and compensation were done on single-stained controls, and cells were sorted into collection tubes and immediately plated at a dilution of 0.5 cells per well into a 96-well plate. Single-cell clones were then expanded and assessed for EMT characteristics.
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