Cells (10,000) were plated per well of a CellCarrier 96-well collagen-coated plate (PerkinElmer), followed by immunostaining for NF-κB p65 or ICAM-1. Plates were scanned, and images were collected with Operetta HTS imaging system (PerkinElmer) at ×20 magnification with 13 fields of view per well, equivalent to between 800 and 1000 cell events. Images were then analyzed with Harmony software (PerkinElmer). Data are means of triplicates.
Operetta hts imaging system
Manufactured by PerkinElmer
Sourced in United States
The Operetta HTS imaging system is a high-content screening (HCS) platform designed for cell-based assays. The system combines automated imaging, image analysis, and data management capabilities to enable efficient and comprehensive analysis of cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Harmony 3, by PerkinElmer (2 mentions)
Columbus 2, by PerkinElmer (2 mentions)
Harmony 4, by PerkinElmer (2 mentions)
Harmony software, by PerkinElmer (1 mentions)
Columbus software, by PerkinElmer (1 mentions)
Paraformaldehyde (pfa), by Avantor (1 mentions)
Imagexpress pico, by Molecular Devices (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Toto 3 iodide, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Avantor (1 mentions)
Click it plus opp protein synthesis assay, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (1 mentions)
Fluozin 3 am, by Thermo Fisher Scientific (1 mentions)
Hoechst nuclear stain, by Thermo Fisher Scientific (1 mentions)
N n n n tetrakis 2 pyridylmethyl ethane 1 2 diamine, by Merck Group (1 mentions)
13 protocols using operetta hts imaging system
1
Quantifying NF-κB and ICAM-1 in Cells
2
Cocultured Splenocyte Interaction Assay
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Cells were cultivated into 96-well plates. Plates were seeded with a total of 1,400 adherent B16-derived cells that harbored GFP and immunomodulators, as indicated. After the cells were incubated for 24 h, 1,400 freshly isolated splenocytes, CD4, or CD8 cells were added, as indicated.
The cocultured cells were incubated with CO2 at 37°C for the indicated times. The CM was removed from the plates and the cells were then fixed in 4% paraformaldehyde and stained with DAPI (Sigma). The plates were scanned (27 fields-of-view/well) using an Operetta HTS imaging system (PerkinElmer) equipped with a 20× air objective lens. The excitation channels 360–400 and 460–490 and the emission filter channels 410–480 were used for eGFP, while 500–550 was used for DAPI. The images were then analyzed using Columbus (version 2.4.0 PerkinElmer).
The cocultured cells were incubated with CO2 at 37°C for the indicated times. The CM was removed from the plates and the cells were then fixed in 4% paraformaldehyde and stained with DAPI (Sigma). The plates were scanned (27 fields-of-view/well) using an Operetta HTS imaging system (PerkinElmer) equipped with a 20× air objective lens. The excitation channels 360–400 and 460–490 and the emission filter channels 410–480 were used for eGFP, while 500–550 was used for DAPI. The images were then analyzed using Columbus (version 2.4.0 PerkinElmer).
3
Cytotoxic Effects of Extracellular Vesicles on Melanoma
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In this assay, B16F10 tumor cells were incubated with splenocytes and EV preparations. The rational of this experiment was that the EV preparation could induce T cell cytotoxicity resulting in a reduced number of B16F10 cells. Briefly, 1,000 B16F10 cells per well were seeded in 96-well plates, adding same numbers of C57BL/6 splenocytes and EVs preparations at specific concentrations. After 72 h of incubation, the splenocytes were removed and the adherent tumor cells were fixed with 4% paraformaldehyde and stained with DAPI (Sigma). Alive B16F10 tumor cells were analyzed by counting the nuclei stained with DAPI, using as template the wells with tumors cells and media without splenocytes. The quantification was performed by the Operetta HTS Imaging System (PerkinElmer). Each well was evaluated in 75 different fields using 20X magnification. Excitation and emission spectra for DAPI was 460–490 nm and 500–550 nm, respectively. All images were analyzed using the Columbus software (version 2.4.0 PerkinElmer) and representative images were processed with the bio-formats plugin in FIJI.
4
Evaluating Cellular Adhesion with hH-EVs
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To evaluate whether hH-EVs could affect cellular adhesion, suspensions containing 5 × 103 cells plus 10 μg/mL hH-EVs were plated onto 96-well plates and incubated for 20 min at 37 °C and 5% CO2. Immediately thereafter, the plates were subjected to 100 rpm agitation to promote nonadherent cell removal. The remaining cells were fixed with 4% paraformaldehyde and stained with 0.05 μL/mL DAPI (4′,6- diamidino-2-phenylindole). The quantification of the adhered cells was performed by an Operetta HTS imaging system (PerkinElmer, Waltham, MA, USA) at 10× magnification with 9 fields of view.
5
EGF-induced pERK Activation Assay
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Cells were plated into 96 well plates (1 × 105 cells/ml for HEK293 cells, 2–8 × 104 cells/ml for MEFs) and incubated at 37 °C, 5% CO2 for 24 before transfection with pCMV6-Affimer-tGFP plasmids using Lipofectamine 2000 as per the manufacturer’s instructions. After a further 24 h cells were serum-starved for 1–18 h before stimulation with EGF (25 ng/ml) for 5 min, rinsed with PBS, and fixed in 4% paraformaldehyde (VWR) for 15 min. Cells were rinsed with PBS and permeabilized with methanol at −20 °C for 10 min, before rinsing with PBS and blocking (1% milk (SigmaAldrich) in PBS) and incubating with anti-pERK antibody (1:150 Cell Signaling Technology 4370) in blocking solution for 1 h at room temperature followed by 3x PBS rinses and incubating with Hoechst 33342 (1 µg/ml Molecular Probes) and anti-rabbit AlexaFluor 546 or 568 (1:1000 Molecular Probes) in blocking solution for 1 h at room temperature. Following a final set of PBS washes, plates were scanned and images collected with an Operetta HTS imaging system (PerkinElmer) or ImageXpress Pico (Molecular Devices) at ×20 magnification. Images were analyzed with Columbus 2.7.1 (PerkinElmer) or MetaExpress 6.7 (Molecular Devices) software.
6
Immunofluorescent Staining of Assay Plates
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Immunofluorescent staining of assay plates was carried out as follows. Media was discarded and cells rinsed in phosphate buffered saline (PBS), before fixation in 4% paraformaldehyde (SigmaAldrich) for 15 mins. Cells were permeabilized with 0.1% Triton X-100 (VWR, Lutterworth, UK) in PBS for 5 mins, cells were then rinsed in PBS and blocked in 1% milk (Marvel, Premier Foods, St Albans, UK) for 5 mins before the addition of mouse anti-Histone H3 (phospho S10, 1∶4000; Abcam ab14955; Cambridge, UK) diluted in 1% milk for 1 hr at room temperature. Following PBS rinses, cells were incubated at room temperature for an hour in the dark with 1% milk containing goat anti-mouse AlexaFluor 568 (1∶1000; Molecular Probes, Eugene, OR) 1 µg/mL DAPI (Molecular Probes) and 500 nM TOTO-3 iodide (Molecular Probes). Following a final set of PBS washes, plates were scanned and images collected with an Operetta HTS imaging system (PerkinElmer) at 20× magnification with 12 fields of view (510×675 µm)/well. Images were then analysed with Columbus 2.2 (PerkinElmer).
7
Quantifying Protein Synthesis in Cells
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Protein synthesis was measured using Click- iT® Plus OPP Protein Synthesis Assay (Molecular Probes, Grand Island, NY). Cells on D0 (hESC) or D15 (cardiomyocytes) were seeded in matrigel-coated 96-well plates, and after 48 h, the staining and detection was performed following the manufacturer’s instructions. The quantitative analysis was performed using an Operetta HTS imaging system (PerkinElmer, Waltham MA, USA). Images of 25 fields per well were evaluated with Harmony 3.5.2 software (PerkinElmer). Fluorescence intensities were measured at the cytoplasm regions around the nucleus. For each condition, 1400 cells were randomly chosen for intensity analysis.
8
Lentiviral-Mediated Chromatin Remodeler Knockdown for 3TF Reprogramming
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TTFs were transduced with lentivirus carrying shRNA targeting specific chromatin remodelers, and re-seeded to 24-well plate at the density of 15 000 cells per well followed by transfection with 3TF lentiviruses. p-ATM activation was analyzed by immunofluorescence quantification 48 h after 3TF transfection. Plate scan and image collection were performed on the Operetta HTS imaging system (PerkinElmer) at 20× magnification with 15 fields of view, Images were then analyzed with Harmony (PerkinElmer). iHep cell colony numbers were counted at day 8 after 3TF transduction.
9
Quantitative Analysis of Osteogenic Cell Adhesion
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After induction of osteogenic differentiation for 24 h, cells were trypsinized, plated on 24-well plates (2,5 × 104 cells/well) and kept for 10, 20 and 40 min in a humidified incubator at 37 °C with 5% CO2 as previously described56 (link). The non-adherent cells were removed by washing twice with PBS. Adherent cells were fixed with 4% paraformaldehyde followed by DAPI staining for counting. The quantitative analysis was performed using an Operetta HTS imaging system (PerkinElmer, Waltham MA, USA) at 100× magnification. Sixty five fields were analyzed and photographed for counting labeled cells with Harmony 3.5.2 software (PerkinElmer).
10
Zinc-Induced Cellular Response Imaging
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After overnight culture, cells were removed from culture plates and transferred to 1.5-ml microcentrifuge tubes. Cells were incubated with 1 μM Fluozin3-AM (Molecular Probes, Eugene, OR) in Live Cell Imaging solution (Molecular Probes) for 1 h at 32°C. Following incubation and washing, cells were resuspended in imaging solution containing Hoechst nuclear stain (2,5′-bi-1H-benzimidazole, 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]; Invitrogen, Burlington, ON, Canada) and incubated for 5, 15, 30, or 45 min at 32°C with 100 μM ZnSO4. After 45 min, TPEN (500 μM N,N,N′,N′-tetrakis[2-pyridylmethyl] ethane-1,2-diamine; Sigma Chemical) was added for 30 min to chelate zinc. Propidium iodide (PI; Invitrogen) was used to evaluate cell viability. After a second wash in imaging solution, cells were transferred to a 96-well cell carrier (PerkinElmer, Woodbridge, ON, Canada) plate with an optically clear bottom. The plate was spun down at 1000 rpm at 4°C for 5 min and immediately scanned (Operetta HTS imaging system; PerkinElmer) at 20× magnification, with 15 fields of view per well. Image analysis software (Columbus version 2.2; PerkinElmer) was used to quantify the mean fluorescent signals from individual cells in each well.
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