Phosphatidylserine externalization was detected using an Annexin V/propidium iodide (PI) staining kit (Life Technologies) following the manufacturer’s instructions. Annexin V/PI positive cells were detected using a Beckman Coulter Gallios 561 Flow Cytometer.
Gallios 561 flow cytometer
Manufactured by Beckman Coulter
The Gallios 561 Flow Cytometer is a high-performance instrument designed for flow cytometry analysis. It utilizes a 561 nm solid-state laser to excite fluorescent dyes and markers, enabling the detection and analysis of multiple cellular parameters simultaneously. The Gallios 561 provides accurate and reliable data for a wide range of applications in biological and medical research.
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6 protocols using gallios 561 flow cytometer
1
Annexin V/PI Apoptosis Assay
2
Fluorometric Substrate Cleavage Assay
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Following drug treatment, PEO1-OR cells were incubated with 33 μM C12FDG (5-dodecanoylaminofluorescein d-β-d -galactopyranoside, Cayman 25583) for 1 h at 37 °C. Fluorescent cleaved substrate was detected using a Beckman Coulter Gallios 561 Flow Cytometer.
3
Cell Cycle Analysis by Flow Cytometry
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PEO1-OR cells were incubated with the indicated concentrations of olaparib and/or UNC0642 for 72 h, then collected by trypsinization and washed once with PBS. Cells were then resuspended and fixed in ice cold 70% ethanol for 1 h at − 20 °C. Cells were washed once with PBS, then resuspended in 200 μL PI staining solution [1× PBS, 50 μg/mL RNase A (Thermo Scientific #EN0531), 50 μg/mL propidium iodide (Thermo Scientific #P3566)] for 30 min at 37 °C, then analyzed by flow cytometry using a Beckman Coulter Gallios 561 Flow Cytometer.
4
Propidium Iodide Cell Staining
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Cells were fixed in ice-cold 95% ethanol for 30 min at 4°C, washed once with phosphate-buffered saline (PBS), and stained with propidium iodide (50 μg/ml) in PBS containing ribonuclease A (50 μg/ml). Further, the cells were incubated at 37°C for 20 min and analyzed on a Gallios 561 flow cytometer (Beckman Coulter) using Kaluza software (Beckman Coulter).
5
Double-Strand Break Repair Assay
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The NHEJ and HR reporter plasmids, pCBASceI (I-SceI expressing plasmid), and pDsRed2-N1 were kindly provided by Dr Vera Gorbunova (University of Rochester, NY). The experiment was done as previously described (24 (link)). To generate reporter cell lines, the linearized NHEJ or HR reporter cassettes (0.5 μg) were transfected into ParC5 cells using Amaxa Nucleofector 2B (Basic Epithelial Cells Kit, Program T-020, Lonza, Walkersville, MD). Post 24 h transfection, cells were selected with 100 μg/ml G418 for 7–10 days to generate stable ParC5-NHEJ and ParC5-HR reporter lines. For the DSB repair assay, the ParC5 reporter lines were pretreated with DMSO, 50 nM dasatinib, or 10 μM imatinib for 24 h prior to cotransfection with 5 μg I-SceI to induce DSBs and 0.1 μg pDsRed2-N1 to normalize for the differences in transfection efficiency. Cells were treated with DMSO, 50 nM dasatinib, or 10 μM imatinib and allowed to repair for 72 h. Cells were analyzed using a Gallios 561 flow cytometer (Beckman Coulter, Indianapolis, IN) and Kaluza software (Beckman Coulter). The repair efficiency was calculated by the ratio of GFP+ cells to DsRed+ cells.
6
Quantifying NHEJ and HDR Efficiencies
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Two-plasmid functional assays were performed to assess distal non-homologous end joining and homology directed repair. Cells were stably transfected with pimEJ5GFP (NHEJ) or pDRGFP (HR) by maintenance in 0.5 μg/mL puromycin and subsequently transfected with I-SceI restriction enzyme. After 72 h, transfected cells were collected and examined using a Beckman Coulter Gallios 561 flow cytometer (Flow Cytometry Shared Resource, University of Colorado) to quantify GFP positive cells. pimEJ5GFP (Addgene #44026) was a gift from Jeremy Stark. pDRGFP (Addgene #26475) and pCBASceI (Addgene #26477) were gifts from Maria Jasin. To control for I-SceI transfection efficiency, DNA was isolated from cells remaining after flow analysis and qPCR was performed using primers specific for transfected I-SceI DNA. Primers for Claudin 4 (CLDN4) gDNA were used as a DNA loading control. Primer sequences are listed in Additional file 4 : Table S2.
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