Cell cycle and subG1 analysis by propidium-iodide staining was performed as previously described (24 (link)). Briefly, cells were fixed in 70% ethanol overnight and stained with 10 μg/ml propidium iodide supplemented with 100 μg/ml RNase A. Cells were analyzed on an Accuri C6 cytometer (BD Biosciences) and cell cycle profiles evaluated by ModFit (Verity Software House). For cell death measurement by DIP analysis, cells were simultaneously fixed and stained in 1 μg/ml DAPI in methanol for 15 min at −20°C. After automated imaging on the BD Pathway 855 microscope (BD Biosciences), cell death was quantified by the DAPI intensity per pixel (DIP) on a single-cell level using AttoVision software (BD Biosciences) (see Supplementary Figure S4).
Pathway 855 microscope
Manufactured by BD
The BD Pathway 855 is a high-performance automated fluorescence microscope designed for cell-based assays and imaging applications. It features automated sample loading, image acquisition, and data analysis capabilities to facilitate efficient and reliable experimental workflows.
Automatically generated - may contain errors
Lab products found in correlation
Attovision, by BD (3 mentions)
Mouse anti brdu antibody, by BD (2 mentions)
Attovision software, by BD (1 mentions)
Accuri c6 cytometer, by BD (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Media Cybernetics (1 mentions)
Image pro software, by Media Cybernetics (1 mentions)
384 well plate, by Greiner (1 mentions)
Imagexpress micro xl, by Molecular Devices (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Anti brdu antibody, by BD (1 mentions)
8 protocols using pathway 855 microscope
1
Cell Cycle and Cell Death Analysis
2
Epigenetics Compound Screening in Chondrosarcoma
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The chondrosarcoma cell lines CH2879, JJ012, and SW1353 were seeded in optimized cell densities (5000, 3000, and 3000/well, respectively) in black 96-well plates. After overnight attachment, cells were treated with the epigenetics compound library at a concentration of 2 µM. After 72 h of treatment, cells were fixed with 4% formaldehyde and stained with 2 µg/mL Hoechst 33342 (H1399, Invitrogen Life-Technologies). Plates were imaged using a BD Pathway 855 microscope (BD Biosciences, Breda, The Netherlands) and the number of Hoechst 33342 positive nuclei was quantified using the Image-Pro software (Media Cybernetics, Rockville, MD, USA). All plates were included in the analysis and data were normalized to the negative controls (i.e., PBS and DMSO) to obtain percent of control values. All compounds that reduced the nuclei count ≥50% in at least one of the cell lines were selected for the secondary epigenetics compound screen. The screen was performed in triplicate. A schematic overview of the primary epigenetics compound screen can be found in Figure 2 A. A list of all compounds, including the numbering and specific targets, can be found in Table S3 .
3
Cyst Formation and Screening Assay
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Cyst culture and screening were performed as described previously (Booij et al., 2017 ). Briefly, cryopreserved mIMCD3 shPkd1 or mIMCD3 Pkd1−/− cells were quick-thawed and grown in culture medium (DMEM/F12 Ham’s, supplemented with FBS and antibiotics) for 72 h. Cells were trypsinized and mixed with Cyst-Gel (OcellO) and plated in 384-well plates (Greiner Bio-One). After addition of culture medium, cysts were allowed to form during 72 h (shPkd1) or 96 h (Pkd1−/−) and subsequently exposed to forskolin (Calbiochem) and test compounds for 72 h. Cysts were thereafter fixed and simultaneously stained for F-actin and nuclei for 12–24 h at 4°C. After washing in 1× PBS (Sigma Aldrich) for 24 h, plates were imaged using either a BD Pathway 855 microscope (BD Bioscience) or ImageXpress Micro XLS (Molecular Devices), with a 4× objective.
4
Mitochondrial Morphology and Membrane Potential
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Cells were seeded and matured as described above in black/clear bottom flat 96-wells plates, after which mitochondrial morphology and membrane potential were assessed microscopically. Cells were washed once in HBSS, subsequently fluorescently stained with 25 nM tetramethylrhodamine methyl ester (TMRM) and co-stained with 0.75 µg/mL Hoechst 33342 in HBSS for 30 min at 37 °C, followed by image acquisition (40 × objective) and 12 × 12 montage using Becton Dickinson (BD) Pathway 855 microscope (BD Bioscience, Breda, The Netherlands), as described before (Iannetti et al. 2016 (link)). Acquired images were processed and quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA), as described previously (Iannetti et al. 2016 (link)). Average data was obtained in N = 3 experiments, consisting of 20 replicates each, and 8 cells/well were analyzed.
5
Annexin-V Apoptosis Assay in 96-well Plates
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Cells were seeded overnight in 96-well μCLEAR plates (Corning) at appropriate densities, then treated with drug solutions at indicated concentrations. At 24, 48 or 72 h post-treatment, cells were stained with Hoechst 33258 (1:10,000) and Annexin-V (1:1000) for 45 min at 37 °C, 5% CO2 before being imaged using BD Pathway 855 Microscope (BD Biosciences). Annexin-V staining was quantified using Cell Profiler software.
6
BrdU Comet Assay for Newly Synthesized DNA
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The BrdU comet assay was performed to detect newly synthesized single strand DNA as previously described (42 (link)). Cells were labeled with 100 μM BrdU for 30 min. BrdU labeled cells were washed with PBS-, placed into fresh medium and incubated for an additional 30 min, 1 h or 2 h to mature replicating DNA strands. Labeled cells were collected, suspended in low-melting-point agarose and added to agarose-coated CometSlides. Slides were incubated in an alkaline lysis solution according to the manufacturer's protocol (Trevigen, 4250-050-K). Following electrophoresis, slides were neutralized by treatment with 0.4 M Tris–HCl, washed with PBS-, and immunostained. Incorporated BrdU substituted newly replicated DNA was detected with an anti-BrdU antibody (BD BioScience, 555627), followed by anti-mouse IgG Alexa 488 (Invitrogen, A-11029). DNA was counter-stained with DAPI and slides were dried according to the manufacturer's instructions (Trevigen, 4250-050-K). Digital images were acquired using a 20× objective lens on a BD pathway 855 microscope, which was controlled by AttoVision (Becton Dickinson). Images were analyzed by CometScore software (TriTeK).
7
BrdU Comet Assay for DNA Damage
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The BrdU comet assay was performed as previously described 43 (link). Briefly, HCT116 and Mus81−/− HCT116 cells were pulsed with 100 μM of BrdU for 30 minutes, then harvested immediately (0h) or chased for 1h or 6h in pre-warmed fresh medium without BrdU. Cells were collected and the cell pellet was resuspended in cold PBS. An alkaline assay was performed according to the manufacture’s instruction (Trevigen, 4250-050-K). Following electrophoresis, the gels were neutralized by 0.4 M Tris.HCl and washed with PBS prior to immunostaining. The BrdU labeled nascent DNA were detected by mouse anti-BrdU antibody (Becton Dickinson, cat.347580, 1:10), then goat anti-mouse IgG conjugated with Alexa 488 (Invitrogen, A-21121, 1:100). The gels were then counterstained with DAPI. The samples were then dried according to the manufacture’s instruction (Trevigen, 4250-050-K). Images were taken by a BD pathway 855 microscope controlled by AttoVision (Becton Dickinson) with 10X or 20X objective lens. Images were analyzed by CometScore software (TriTeK).
8
BrdU Comet Assay for DNA Damage
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