Monolayer cells or 4-day-cultured mammospheres were trypsinized and dissociated into single-cell suspensions. ALDHhi BCSCs were isolated with the ALDEFLUOR kit according to the manufacturer’s protocol (STEMCELL Technologies, no. 01700), followed by staining with PI (1 μg/mL, Sigma-Aldrich) for 15 minutes at 25°C. The percentage of PI-positive BCSCs was examined on a FACSCanto RUO cell analyzer (Becton Dickinson).
Facscanto ruo cell analyzer
Manufactured by BD
The FACSCanto RUO cell analyzer is a flow cytometry instrument designed for research purposes. It provides the capability to analyze and sort a wide range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Aldefluor kit, by STEMCELL (2 mentions)
Facsaria 2 sorp cell sorter, by BD (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Cellrox deep red flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Northern lights flow cytometer, by Cytek (1 mentions)
Cellular ros assay kit, by Abcam (1 mentions)
3 protocols using facscanto ruo cell analyzer
1
Isolation and Analysis of ALDH^hi BCSCs
2
Isolating and Characterizing Tumor Cell Populations
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Human PDX tumors, mouse MMTV-PyMT tumors, or MDA-MB-231 and MCF-7 xenograft tumors were crosscut into small pieces in Hanks’ balanced salt solution (HBSS; Sigma-Aldrich), washed, and digested for 45 min with collagenase/hyaluronidase/DNase (Sigma-Aldrich) in a 37°C shaker. Cultured cells were trypsinized and dissociated into single-cell suspensions. Cells were stained for 30 min with fluorescent-conjugated antibodies listed in table S2 on ice, followed by washing with HBSS and centrifugation at 500g for 5 min. Cells were examined on a FACSCanto RUO Cell Analyzer (Becton Dickinson) or a CytoFLEX Flow Cytometer (Beckman Coulter) or sorted on a FACSAria II SORP Cell Sorter (Becton Dickinson). Aldefluor assay was performed with an ALDEFLUOR kit according to the manufacturer’s protocol (STEMCELL Technologies).
3
Measuring Cellular ROS in BCSC Subpopulations
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Cellular ROS in spheres was measured with a cellular ROS assay kit (Abcam, no. ab113851) according to the manufacturer’s protocol. The fluorescence of 2′,7′-dichlorofluorescein was normalized to cell number. Cellular ROS in ALDHhi BCSCs and ALDHlo non-BCSCs was measured with a CellROX deep red flow cytometry assay kit (Invitrogen, no. C10491) according to the manufacturer’s protocol. Cells were examined on a FACSCanto RUO cell analyzer (Becton Dickinson) or Cytek Northern Lights flow cytometer (Cytek Biosciences).
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