Cells were seeded onto 18mm glass coverslips in 12-well plates. The following day, cells were treated with DMSO or 2 μM Torin-1 for 3 h prior to processing for microscopy. Briefly, cells were washed with phosphate buffered saline (PBS) then fixed for 15min at RT with PBS containing 4% (v/v) paraformaldehyde. After fixation, cells were washed with PBS, blocked and permeabilized for 1 h with PBS containing 5% (v/v) goat serum and 0.1% (v/v) Triton X-100 then incubated overnight at 4°C in PBS containing 5% (v/v) goat serum and mouse anti-FLAG antibody (1/1000). The following day, cells were washed with PBS, incubated for 1 h at RT with goat anti-mouse secondary antibody then washed and mounted on glass slides. Confocal images were captured with an Andor Zyla 4.2 plus digital camera at 40× using a Nikon Ti2-E inverted microscope with W1 spinning disk confocal and Nikon NIS-Elements. For LysoView 488, DQ-BSA or MitoTracker Red, cells were stained as per manufacturer’s instructions, and live confocal images were captured using a Perkin Elmer Opera Phenix system equipped with a high NA 20x air objective and Perkin Elmer Harmony High-Content Imaging and Analysis Software.
Opera phenix system
Manufactured by PerkinElmer
Sourced in Japan
The Opera Phenix system is a high-content screening platform designed for cellular imaging and analysis. It provides automated imaging and data analysis capabilities for a variety of cell-based assays. The system integrates components for sample handling, image acquisition, and data processing to enable efficient and standardized workflows for drug discovery and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescently conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
D9663, by Merck Group (3 mentions)
Dapi nuclear stain, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde pbs, by Thermo Fisher Scientific (2 mentions)
Axioobserver epi fluorescent microscope, by Zeiss (2 mentions)
Methanol, by Fujifilm (2 mentions)
Blocking one, by Fujifilm (2 mentions)
Alexa fluor 488 conjugated anti mouse rabbit, by Thermo Fisher Scientific (2 mentions)
Bz 710x, by Keyence (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ti2 e inverted microscope, by Nikon (1 mentions)
Harmony high content imaging and analysis software, by PerkinElmer (1 mentions)
Nis element, by Nikon (1 mentions)
Click it edu alexa fluor 647 kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Imagexpress micro imaging system, by Molecular Devices (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
11 protocols using opera phenix system
1
Immunofluorescence Microscopy of Torin-1 Treatment
2
Immunofluorescence Staining of 2D Cell Cultures
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For 2D cell cultures, cells were fixed with 3% paraformaldehyde (Thermo Fisher Scientific) in PBS for 20 min at room temperature and subsequently washed three times with PBS, followed by blocking and permeabilization with 5% donkey serum (D9663, Sigma-Aldrich) and 0.3% Triton X-100 (Fisher Scientific) in PBS (PBS-DT) for 1 h. Cells were incubated with primary antibodies (Table S2 ) at 4°C overnight, then washed three times with PB, and incubated with fluorescently conjugated secondary antibodies (Invitrogen) at 1:250 dilution for 1 h at room temperature. Both primary and secondary antibodies were diluted in PBS-DT. Cells were washed with PBS and stained with 0.1 µg ml−1 DAPI nuclear stain (Thermo Fisher Scientific) before imaging.
Cell proliferation analysis with EdU was performed by treating cells with 10 µM EdU for 20 min before fixation. Staining for EdU was performed using a Click-iT EdU Alexa Fluor 647 kit (Invitrogen) following manufacturer specifications.
Confocal imaging was performed on a Perkin Elmer Opera Phenix system (QB3 High-Throughput Screening Facility). Bright-field and wide-field fluorescence imaging was performed on a Zeiss AxioObserver epi-fluorescent microscope and a Molecular Devices Image Xpress Micro imaging system (CIRM/QB3 Shared Stem Cell Facility).
Cell proliferation analysis with EdU was performed by treating cells with 10 µM EdU for 20 min before fixation. Staining for EdU was performed using a Click-iT EdU Alexa Fluor 647 kit (Invitrogen) following manufacturer specifications.
Confocal imaging was performed on a Perkin Elmer Opera Phenix system (QB3 High-Throughput Screening Facility). Bright-field and wide-field fluorescence imaging was performed on a Zeiss AxioObserver epi-fluorescent microscope and a Molecular Devices Image Xpress Micro imaging system (CIRM/QB3 Shared Stem Cell Facility).
3
Cytotoxicity Assay with Opera Phenix
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The Opera Phenix system (Perkin Elmer) was also used to follow both target cell growth and killing simultaneously. In this assay, an effector: target ratio of 4:1 was used. Target cells were counted and plated at 15 000 cells/well in 100 uL/well (CAMA-1-A2B2M) on the day prior to setting up the assay peripheral blood mononuclear cells were thawed from liquid nitrogen on day 2 counted, stained with 2 uM CellTracker DeepRed and plated in 50 uL/well. NucView488 reagent was added to effectors to give a 0.8 uM final concentration on the well. Hoechst was added to effectors to give a final concentration of 150 nM in the well. Plates were imaged with the 5× objective every 6 hours over 96 hours.
4
Irofulven Cytotoxicity Screening in hTERT Fibroblasts
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BJ1/hTERT (hTERT-immortalized normal human foreskin fibroblast) cells were seeded in 384-well plates (CellCarrier-384 Ultra; PerkinElmer) and transfected with siRNAs (silencer select, pool of three siRNAs per one gene, final siRNA concentration: 3 nM per well; Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). 24 h after transfection, cells were treated with either: (1) 2 μg/ml irofulven for 1 h and cultured for another 4 d without irofulven or (2) with 75 ng/ml irofulven for 4 d. Cells were fixed with 4% paraformaldehyde and stained with 1 μg/ml Hoechst 33342 (Thermo Fisher Scientific) in PBS. The proportions of stained cells were determined using an Opera Phenix system and Harmony software (PerkinElmer).
5
Cellular Internalization Imaging Assay
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Cells were plated into poly-D-lysine–coated 96-well cyclic olefin imaging microplates (PerkinElmer). Internalization assays were performed as described above. At the end of the study, cells were fixed with 4% paraformaldehyde for 10 minutes, washed in PBS, and blocked in PBS plus 2% BSA for 45 minutes. Internalization was performed as described above, and cells were then fixed with 4% paraformaldehyde and washed with PBS. Nuclear staining was performed by incubation with Hoechst 33342 stain (0.5 μg/mL) in PBS for 5 minutes. Cells were imaged using the Opera Phenix system (PerkinElmer) by spinning-disk confocal fluorescence imaging with a water-immersion 20× objective and appropriate channels for GFP (excitation, 499 nm; fluorescence, 520 nm) and Hoechst (excitation, 350 nm; fluorescence, 461 nm).
6
Immunofluorescence Staining Protocol
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Cells were fixed with 3% PBS – paraformaldehyde (ThermoFisher) for 20 min at room temperature and subsequently washed three times with PBS. Blocking and permeabilization was done with 5% donkey serum (D9663, Sigma-Aldrich) and 0.3% Triton X-100 (Fisher Scientific) (PBS-DT) for 1 hour. Cells were incubated with primary antibodies (Table S1 ) at 4°C overnight, then washed three times with PBS, and incubated with fluorescently conjugated secondary antibodies (Invitrogen) at 1:250 dilution for 1 hour at room temperature. Both primary and secondary antibodies were diluted in PBS-DT. Cells were washed with PBS and stained with 0.1 μg mL −1 DAPI nuclear stain (ThermoFisher) prior to imaging. Confocal imaging was performed on a Perkin Elmer Opera Phenix system (QB3 High-Throughput Screening Facility). Brightfield and widefield fluorescence imaging was performed on a Zeiss AxioObserver epi-fluorescent microscope (CIRM/QB3 Shared Stem Cell Facility).
7
Uptake Kinetics of Engineered EVs
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Acceptor cells were cultured following standard culturing conditions. Cytoplasm was stained with ViaFluor®488 proliferation dye (Biotium, Fremont, CA, USA) 24h prior to the uptake experiment and seeded at 3 x 103 cells per well in a 384-well plate (PerkinElmer, Waltham, MA, United States). The following day, cells were stained with NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342) (Thermo Fisher Scientific), washed and maintained in regular growth medium. Langendorff perfusate-derived EVs were stained with DiOC18(7) (DiR) (Thermo Fisher Scientific). Stained EVs were added to cell medium at a final concentration of 13 ng protein/μl. Cells were imaged for 16 h using an Opera Phenix™ system (PerkinElmer). Analysis of internalization of EVs was performed using Harmony v.4.9 (Perkin Elmer).
8
Immunofluorescence Staining of Myosin Subtypes
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Cells were fixed with 2% paraformaldehyde (PFA)/PBS (Wako), methanol (Wako), blocked with Blocking One (Nacalai) for 45 min, and subsequently incubated with primary antibodies diluted in 5% Blocking One/ PBST (Wako) at 4°C overnight. Cells were washed three times in PBS and incubated with secondary antibodies diluted in 5% Blocking One/PBST for 1 h at room temperature. DAPI (Sigma) was used to counterstain the nuclei. Samples were visualized and photographed with BZ-710X (Keyence, Osaka, Japan) or the Opera Phenix System (PerkinElmer, Waltham, MA). The antibodies used for this study were as follows: mouse anti-myosin heavy chain (pan-MHC) monoclonal (MF20; 1:500, R&D, Minneapolis, MN), mouse anti-skeletal myosin (FAST/MYH1&2), monoclonal (MY-32; 1:100, Sigma), mouse anti-Myosin-2 (MYH2) monoclonal (MABT840; 1:100, Millipore, Burlington, MA), rabbit anti-MYH3 polyclonal (1:100, Atlas antibodies, Bromma, Sweden), mouse anti-MYH7 monoclonal (A4.840; 1:200, Santa Cruz, Dallas, TX), rabbit anti-MYH8 polyclonal (1:100, Novus Biologicals, Littleton, CO), rabbit anti-sarcomeric α actinin polyclonal (1:500, Abcam, Cambridge, UK), mouse anti-dystrophin (Rod domain) monoclonal (DYS1; 1:20, Leica, Buffalo Grove, IL), Alexa Fluor 488-conjugated anti-mouse/rabbit, and Alexa Fluor 647-conjugated anti-mouse/rabbit antibodies (1:500, Invitrogen).
9
Phenotypic Screening of T. cruzi Infection
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The Perkin Elmer Opera Phenix™ System was used for phenotypic screening. For this system, 60,000 HG39 cells were plated on 96-well plates. The cells were infected with T. cruzi Tulahuen (MOI = 10) for 24 hours, followed by washing and subsequent treatment with carvedilol (10 µM) in RPMI medium for 24, 48 or 72 h, with daily medium change. Control cells were maintained in RPMI medium plus an equal volume of carvedilol vehicle (DMSO). Cells were fixed with 4% paraformaldehyde, and immune staining was performed with mouse anti-Leishmania HSP90 serum (Ommen et al., 2010 (link)) (1: 5000) and Alexa Fluor647 anti-mouse (1: 8000) + DAPI (1: 100). The numbers of parasites under each condition were quantified in the Opera Phenix System (Bea, 2002 (link)).
10
Multicolor Immunofluorescence Imaging of Skeletal Muscle Proteins
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The cells were fixed with 2% paraformaldehyde (PFA)/PBS (Wako) and methanol (Wako), blocked with Blocking One (Nacalai) for 45 min, and subsequently incubated with primary antibodies diluted in 5% Blocking One/PBST (Wako) at 4 °C overnight. The cells were washed in PBS and incubated with secondary antibodies diluted in 5% Blocking One/PBST for 1 h at room temperature (24–26 °C). Then, 4′,6-diamidino-2-phenylindole (DAPI; Sigma) was used to counterstain the nuclei. The samples were visualized and photographed with a BZ-710X (Keyence; Osaka, Japan) or Opera Phenix System (PerkinElmer; Waltham, MA, USA). The primary antibodies used for this study were: mouse anti-myosin heavy-chain (pan-MHC) monoclonal (MF20; 1:500; R&D; Minneapolis, MN, USA), mouse anti-dystrophin (Rod domain) monoclonal (DYS1; 1:20; Leica; Buffalo Grove, IL, USA), rabbit anti-STIM1 monoclonal (D88E10; 1:800; Cell Signaling Technology; Danvers, MA, USA), and mouse anti-Orai1 monoclonal (G-2; 1:5; Santa Cruz; Dallas, TX, USA) antibodies. Alexa Fluor 488-conjugated anti-mouse/rabbit and Alexa Fluor 647-conjugated anti-mouse/rabbit antibodies (1:500, Invitrogen) were used as secondary antibodies.
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