For all assays, culture plates were fixed with 10% formalin for 10 min at room temperature (RT) and washed 3-times with 1 × PBS. Fixed plates were stained with BODIPY 493/503 (2 µM in PBS) for 1 h RT and washed with PBS. Fixed cells were then incubated with DAPI (0.5 µg/mL in 1 × BD permeabilization solution) for 10 min at RT and washed with PBS prior to imaging. Fluorescent imaging was performed using a Perkin Elmer Operetta CLS high content imager. Images were taken at 20 × (35 fields/well) and 5 × (whole well).
Operetta cls high content imager
Manufactured by PerkinElmer
Sourced in Germany, United States
The Operetta CLS high content imager is a versatile imaging system designed for a range of high-content screening applications. The system captures high-quality images and performs automated image analysis to provide quantitative data on cellular and subcellular features.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Fix perm solution, by BD (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Harmony software 4, by PerkinElmer (1 mentions)
7 protocols using operetta cls high content imager
1
Fluorescent Imaging of Fixed Cells
2
Visualizing SNCA mRNA and protein levels
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RNAscope is a variation of FISH (fluorescent in situ hybridization) used to visualize RNA transcripts within cells. Kits and probes were purchased from ACD (Fluorescent multiplex detection reagent kit, #320851 and SNCA, #571241) and used according to manufacturer’s instructions. Briefly, cells were fixed with 4% PFA, SNCA mRNA was amplified, and the secondary fluorescent detection probe (Thermo Fisher, Alexa Fluor 488 #A11000) was added. Cells then underwent ICC to examine αsyn protein levels. Cells were permeabilized using 0.1% Triton-X in 1X PBS and incubated at RT. Following additional washes, bovine serum albumin (BSA) was used to block non-specific antigens. Primary antibody, 4B12 (αsyn, # 807802, 1:1,000) was diluted in blocking buffer and incubated at RT. Secondary fluorescent antibody Alexa Fluor 568 (Thermo Fisher, # A11004, 1:1,000), were incubated after washes in the dark. Hoechst staining (#H3570, 1:5,000) was completed prior to imaging and quantification on a Perkin Elmer Operetta CLS High Content Imager (Johns Creek, GA).
3
Spheroid Formation and Quantification
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1 × 103 cells were plated in ultralow attachment plates (96-well format, Perkin Elmer) in a final volume of 100 µL. After seeding, plates were centrifuged at 2,500g for 10 minutes at RT and incubated at 37 degrees with 5% CO2. After 24 hours from plating, the extracellular matrix (Geltrex, Thermo Fisher Scientific, cat. no. A1413201 ) was added in a 3% final concentration. Z-stack images of each spheroid were acquired daily starting from 48 hours using the Operetta CLS High Content Imager (Perkin Elmer). The area of spheroids (maximal projection) was quantified using a dedicated script developed with the Harmony software (Perkin Elmer).
4
Quantitative Virus Infectivity Assay
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Control and experimental RVH samples were used to infect Vero cells plated overnight in 96 well plates. A serial dilution of viruses in Vero cell media was performed to obtain a dose response infectivity curve. 24 h post infection, virus containing media was removed and cells were permeabilized and fixed using BD Perm Fix solution directly in 96 well plate. Staining of virus in cells was then performed using anti-VSV rabbit polyclonal antibody (Imanis) at a 1:1000 dilution for one hour at 37degC. Secondary staining was performed with anti-rabbit AF568 secondary at 1:200 dilution for 40 min at 37degC. A DAPI counterstain was applied and plates were imaged using Perkin Elmer Operetta CLS high content imager. Image analysis was performed using the Harmony software (PE). Statistical analysis was done on log transformed data using Graph Pad Prism software. A P value of < 0.05 was considered highly significant. P values between 0.1 and 0.05 were considered moderately significant.
5
Fluorescence Microscopy of Biofilm Viability
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Black 96‐well plates with a glass bottom (PerkinElmer, Hamburg, Germany) were used for fluorescence microscopy. To avoid dehydration of the biofilms, plasma‐treated biofilms were resuspended in 300 µl of 0.85% NaCl after treatment. The LIVE/DEAD BacLight™ Bacterial Viability Kit was used as previously described (2.5). Epifluorescence images were acquired using Operetta CLS High‐Content Imager (PerkinElmer, Hamburg, Germany) using a 5× objective (air, NA = 0.16, Zeiss, Oberkochen, Germany). Depending on the experiment, several fields of view were recorded and combined in the software. SYTO™ 9 was excited by a 475 nm (110 mW) LED, and the fluorescence was collected with a 525 ± 25 nm band‐pass filter. Propidium iodide was excited by a 550 nm (170 mW) LED, and the emission light was collected with a 610 ± 40 nm band‐pass filter. A laser autofocus (785 nm) was available for all measurements. The images were displayed using Harmony 4.6 software.
6
Immunofluorescence Analysis of Podocyte Tight Junctions
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For immunofluorescence experiments, PTL were differentiated in CellCarrier ultra-black 96-well plates. On day 14 of differentiation, PTL were fixed with 4% PFA (20 min), permeabilized with 0.1% Triton X-100 (10 min), and blocked with 5% BSA (1 h). Primary antibodies were applied for 1.5 h at RT in a DPBS 1% BSA solution and include megalin/LRP2 (1:100) (R&D systems MAB9578), ZO-3 (1:1600) (Cell Signaling Technology 3704), and occludin Alexa Fluor 594 (1:250) (ThermoFisher Scientific 331594). Secondary antibodies were applied (when applicable) together with and Hoechst 33342 (1:10.000) (ThermoFisher H3570) for 1 h at RT in a DPBS 1% BSA solution and include α-rabbit Alexa FluorTM 546 (1:1000) (ThermoFisher A10040) and α-mouse Alexa FluorTM 546 (1:1000) (ThermoFisher A10036). Cells were imaged using the Operetta CLS High-Content Imager (Perkin Elmer) with 40 × water objective, and images were exported using the Harmony software 4.8.
7
High-content Imaging of Zebrafish Tumor Footprint
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For automated imaging, larvae were anesthetized in 1x Tricaine and embedded in a 96-well ZF plate (Hashimoto Electronic Industry Co, Japan) with 0.5 % ultra-low gelling agarose (Cat. No.
A2576-25G, Sigma-Aldrich Chemie GmbH, Germany). The Operetta CLS high-content imager (PerkinElmer, USA) was used for image acquisition: 5x air objective, Brightfield (40 ms, 10%), GFP (excitation: 460-490 nm at 100%, emission: 500-550 nm for 400 ms), tagRFP (excitation: 530-560 nm at 100%, emission: 570-650 nm for 400 ms), CellTrace Violet (excitation: 390-420 nm at 100%, emission: 430-500 nm for 600 ms). 23 planes with a distance of 25 µm were imaged per field.
Tumor size was quantified with the Harmony Software 4.9 (PerkinElmer, USA). The area of the tumor projected along the z-axis onto the x-y-plane ("footprint area") was used for further analysis.
A2576-25G, Sigma-Aldrich Chemie GmbH, Germany). The Operetta CLS high-content imager (PerkinElmer, USA) was used for image acquisition: 5x air objective, Brightfield (40 ms, 10%), GFP (excitation: 460-490 nm at 100%, emission: 500-550 nm for 400 ms), tagRFP (excitation: 530-560 nm at 100%, emission: 570-650 nm for 400 ms), CellTrace Violet (excitation: 390-420 nm at 100%, emission: 430-500 nm for 600 ms). 23 planes with a distance of 25 µm were imaged per field.
Tumor size was quantified with the Harmony Software 4.9 (PerkinElmer, USA). The area of the tumor projected along the z-axis onto the x-y-plane ("footprint area") was used for further analysis.
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