For the flow cytometry screening experiment, organoids were grown in 96 well glass-bottom plates (Cellvis) that were pre-cooled and held on ice during seeding. Organoid fragments in Matrigel (50 μl/well) were distributed in pre-cooled plates with an automated pipette, then plates were transferred to a rotary plate shaker for 30 s at 150 rpm, before the Matrigel was solidified at 37°C. With help of a Viaflo 96-channel pipette (Integra Biosciences) 200 μl/well ENR without or with inhibitors were added of which 100 μl were replaced daily during the 96 h time course. To obtain single cells, culture media was removed, 100 μl/well TrypLE express (Thermo Fisher Scientific) added and Matrigel disrupted by repeated pipetting with a multichannel pipette. Staining with Zombie Aqua, CD326-BV421, CD24 -PerCp-Cy5.5, CD44-AF647, CD117-PE-Cy7 (all Biolegend, see Supplementary Table 3 for detailed list), and UEA1-FITC (Invitrogen) was carried out as described above. Samples were run on a MACSQuant X instrument (Miltenyi Biotec) equipped with 405, 488, 647 nm laser lines and analyzed as described above. Euclidean distance clustering tree of normalized median population frequencies was generated with GNU R package ggtree (Yu et al., 2018 (link)).
Macsquant x instrument
Manufactured by Miltenyi Biotec
The MACSQuant X is a flow cytometry instrument designed for high-throughput analysis. It features multiple lasers and detectors for the simultaneous measurement of multiple parameters. The MACSQuant X is capable of performing automated sample processing and analysis, providing researchers with a powerful tool for cell analysis and sorting applications.
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Lab products found in correlation
Cd24 percp cy5, by BioLegend (1 mentions)
96 well glass bottom plate, by Cellvis (1 mentions)
Cd117 pecy7, by BioLegend (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Cd44 af647, by BioLegend (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BioLegend (1 mentions)
7 aad, by BioLegend (1 mentions)
2 protocols using macsquant x instrument
1
Flow Cytometry Screening of Organoid Cultures
2
Annexin V Apoptosis Assay in Cervical Cancer
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Cell apoptosis was evaluated using flow cytometry based on Annexin V staining following the manufacturer's protocol. In brief, cervical cancer cells were seeded at a density of 5 × 106 cells per well in a 6-well plate. After 72 h of incubation with varying concentrations of monensin in RPMI 1640 supplemented with 10% FBS, the cells were detached using trypsin and subsequently stained with FITC Annexin V Apoptosis Detection kit along with 7-AAD (BioLegend). This staining was performed at 4°C in the dark for 30 min, followed by flow cytometry analysis using a MACSQuant X instrument (Miltenyi Biotec).
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