To evaluate the apoptotic rate for PDAC cell lines, 5000 cells/well were seeded in white 96-well plates and cultured for 24 h with Pladienolide-B or vehicle, and apoptotic rates were measured using Caspase-Glo 3/7 Reagent (Promega), following the manufacturer’s instructions [36 (link)]. For Annexin-V staining, floating and attached cells were pooled and resuspended in 1X Annexin-V staining buffer containing Annexin-V-FITC diluted 1:20 (Cat no. 29001, Biotium, Freemont, CA) and then, incubated for 20 min at room temperature prior to flow cytometric analysis. Cytometry data was acquired with an Invitrogen™ Attune™ NxT 4-laser cytometer with software version 3.1.1.
Attune nxt 4 laser cytometer
Manufactured by Thermo Fisher Scientific
The Attune™ NxT 4-laser cytometer is a versatile flow cytometry instrument designed for high-performance analysis and sorting of cells and other biological particles. It features four laser excitation sources and the ability to detect up to 16 fluorescent parameters simultaneously.
Automatically generated - may contain errors
4 protocols using attune nxt 4 laser cytometer
1
Evaluating Apoptosis in PDAC Cell Lines
2
Identification of Pancreatic Cancer Stem Cells
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Primary pancreatic cells (monolayers and spheres) were trypsinized and resuspended in Sorting Buffer (3 μM EDTA, and 3% FBS in 1X PBS). To identify CD133 positive CSC, the following conjugated antibodies were used: anti-CD133/1-APC or PE; (Miltenyi), and appropriate isotype-matched control antibodies. For autofluorescent detection, cells were excited with blue laser 488 nm and selected as the intersection with the filters 530/30 (BL1) and 590/40 (BL2) [20 (link)]. For all assays, 2 mg/mL DAPI (Cat no. D9564, Sigma-Aldrich) was used to exclude dead cells with laser VL1. Data were analyzed with FlowJo 9.3 software (Tree Star Inc., Ashland, OR.). Cytometry data was acquired with an Invitrogen™ Attune™ NxT 4-laser cytometer with software version 3.1.1.
3
Apoptosis Assay for PDAC Cell Lines
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To evaluate the apoptotic rate for PDAC cell lines, 5,000 cells/well were seeded in white 96-well plates and cultured for 24 h with Pladienolide-B treatment or vehicle, and apoptotic rate was measured using Caspase-Glo 3/7 Reagent (Promega), following the manufacturer's instructions (35) . For Annexin-V staining, oating and attached cells were pooled and resuspended in 1X Annexin-V staining buffer containing Annexin-V-FITC diluted 1:20 (Cat no. 29001, Biotium, Freemont, CA) and then, incubated for 20 min at room temperature prior to ow cytometric analysis. Cytometry data was acquired with an Invitrogen™ Attune™ NxT 4-laser cytometer with software version 3.1.1.
4
Isolation and Analysis of Pancreatic Cancer Stem Cells
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Primary pancreatic cells (monolayers and spheres) were trypsinized and resuspended in Sorting Buffer (3 µM EDTA, and 3 % FBS in 1X PBS). To identify CD133 positive CSC, the following conjugated antibodies were used: anti-CD133/1-APC or PE; (Miltenyi), and appropriate isotype-matched control antibodies. For auto uorescent detection, cells were excited with blue laser 488 nm and selected as intersection with the lters 530/30 (BL1) and 590/40 (BL2) (20) . For all assays, 2 mg/mL DAPI (Cat no. D9564, Sigma) was used to exclude dead cells with laser VL1. Data were analyzed with FlowJo 9.3 software (Tree Star Inc., Ashland, OR.). Cytometry data was acquired with an Invitrogen™ Attune™ NxT 4-laser cytometer with software version 3.1.1.
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