CRC Organ Chips were manually washed by flowing PBS through the endothelial and epithelial channels. The chips were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, #15710), incubated for 15 minutes, and permeabilized with 1% saponin. Blocking buffer of 2% bovine serum albumin (BSA) and primary antibodies were incubated overnight at 4°C before a 2 hour incubation with secondary antibodies (1:100, Molecular Probes, Invitrogen, #A21428 and #A21235) diluted in blocking buffer. The primary antibodies used for these studies were anti-E-cadherin (1:25; Abcam, #ab1416), anti-VE-cadherin (1:25; Abcam, #ab33168), anti-vimentin (1:50; Abcam, #ab93547), anti-ZO-1 (1:100; Thermo-Fisher, #339194). DAPI (Sigma-Aldrich, D9542) was used to label all nuclei. Chips were imaged using the Perkin Elmer Operetta CLS High Content System. Quantitation of E-Cadherin+ and Vimentin+ (tumor cells identified by GFP) was performed using the Perkin Elmer Harmony software based on intensity thresholds.
Operetta cls high content system
Manufactured by PerkinElmer
The Operetta CLS High Content System is a high-throughput imaging platform designed for cell-based screening and analysis. It provides automated image acquisition and analysis capabilities, enabling researchers to efficiently study cellular morphology, function, and behavior.
Automatically generated - may contain errors
Lab products found in correlation
Ab1416, by Abcam (2 mentions)
A21428, by Thermo Fisher Scientific (2 mentions)
A 21235, by Thermo Fisher Scientific (2 mentions)
Ab33168, by Abcam (1 mentions)
D9542, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Multiflo fx multi mode dispenser, by Agilent Technologies (1 mentions)
Matlab 2017a software, by MathWorks (1 mentions)
Operetta high content screening system, by PerkinElmer (1 mentions)
Columbus image data storage and analysis system, by PerkinElmer (1 mentions)
Alexa fluor 647 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti γh2ax ser139, by Cell Signaling Technology (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Harmony 4, by PerkinElmer (1 mentions)
Ab113851, by Abcam (1 mentions)
Cellcarrierultra 96, by PerkinElmer (1 mentions)
Ab92547, by Abcam (1 mentions)
9 protocols using operetta cls high content system
1
Immunofluorescent Staining of Organ Chips
2
High-Throughput Cancer Drug Screening
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Cells were seeded in 384-well plates at 1,000 cells/well in 40 µL of growth medium. After 24 hours, cells were treated with varied concentrations of drugs from a small Food and Drug Administration (FDA)-approved cancer drug library consisting of 62 cancer drugs (purchased from Selleck Chemicals or MedChemExpress) with 28 specific targeted molecules for 5 days. Cell numbers were determined by the Operetta® High Content Screening System (PerkinElmer, Waltham, MA, USA). The images were used to calculate growth rate inhibition (GR) values using the following equation (18 (link)).
For high-throughput drug screening, cells were seeded in 384-well plates using a MultiFlo FX Multimode Dispenser (BioTek Instruments, Winooski, VT, USA) at 1,000 cells/well in 40 µl of growth medium. After 24 hours, cells were treated with the drugs in the drug library (at their respective GR75 values) in 20 µl of the total growth medium and using an EpMotion pipetting robot (Eppendorf, Hamburg, Germany). The cells were incubated for 5 days in a 37°C 5% CO2 environment. Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and imaged using an Operetta CLS™ high-content system (PerkinElmer). The nuclei number was analyzed using a Columbus Image Data Storage and Analysis System (PerkinElmer), and plotted using MATLAB 2017a software (MathWorks, Natick, MA, USA).
For high-throughput drug screening, cells were seeded in 384-well plates using a MultiFlo FX Multimode Dispenser (BioTek Instruments, Winooski, VT, USA) at 1,000 cells/well in 40 µl of growth medium. After 24 hours, cells were treated with the drugs in the drug library (at their respective GR75 values) in 20 µl of the total growth medium and using an EpMotion pipetting robot (Eppendorf, Hamburg, Germany). The cells were incubated for 5 days in a 37°C 5% CO2 environment. Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and imaged using an Operetta CLS™ high-content system (PerkinElmer). The nuclei number was analyzed using a Columbus Image Data Storage and Analysis System (PerkinElmer), and plotted using MATLAB 2017a software (MathWorks, Natick, MA, USA).
3
Intracellular Glucose Monitoring using DCFH-DA
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The intracellular glucose was dynamically monitored by using DCFH-DA dye, which could be de-esterified intracellularly to form DCFH. DCFH could react with H2O2 (the product of glucose conversion catalyzed by GOx) and produce highly fluorescent DCF. Cells were primarily allowed to adhere for 24 h in 96-well plates and be washed for three times with glucose-free PBS prior to the detection. Subsequently, the DCFH-DA dye (at a work concentration of 10 µM) and the GOx-aZIF or GOx-ZIF-8 at certain concentrations were simultaneously added into the cells for dynamic detection (4.5, 15, and 45 µg mL−1). The fluorescent images of the cells were recorded during 4-h incubation period (37 °C and 5% CO2) via the “Operetta CLS” High Content System (PerkinElmer). Alex Fluor 488 channel (LED power) was selected to acquire the fluorescent signal from DCF, which was excited at 460–490 nm and recorded at 500–550 nm emission wavelength via standard filter sets. Ten percent power was set for the excitation, and 10 ms exposure time was controlled to avoid saturated pixels. Particularly, the instrument was equipped with a 16-bit sCMOS camera, which operated in a fast acquisition mode for exposure time ≤10 ms. The pinhole size was 55 µm.
4
Immunofluorescent Detection of γH2AX
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The cells were fixed with 4% paraformaldehyde in PBS, washed and permeabilized by 0.1% Triton X-100 in PBS, and non-specific binding was blocked by the Odyssey® blocking buffer. Anti γH2AX, Ser139 (Cell Signaling Technology) was used to detect γH2AX+ cells. Alexa fluor 647 donkey anti-mouse IgG (Thermo Fisher Scientific) was used as secondary antibody. The nuclei were counter-stained using DAPI. Images were acquired using the Operetta CLS™ high-content system (PerkinElmer). At least 300 cells were counted. Positive foci formation of γH2AX, cells were determined by more than 3 foci per cell.
5
Intracellular Trafficking of mRNA Lipoplexes
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DC 2.4 and RD cells (at 50–60% confluency) were transfected with CellLight® Late Endosomes-RFP, BacMam 2.0 (Thermo Fisher Scientific) and incubated overnight at 37 °C in a 5% CO2 environment. Then, cells were treated with the MFP488-labeled DNCA/CLD-mRNA-1096 (MFP488-labeled mRNA-1096 = 1 μg) and incubated for 3 h at 37 °C in a 5% CO2 environment. MFP488-labeled Lipofectamine 2000-mRNA-1096 (MFP488-labeled mRNA-1096 = 1 μg) was set as reference. After removal of the supernatants, cells were washed with PBS, sequentially stained with Lyso-Tracker deep Red (50 nM; Thermo Fisher Scientific) and Hoechst 33342 (1:100; Beyotime) at 37 °C in a 5% CO2 environment for 45 min and 5 min, respectively. After final washes with PBS, cells were observed by Operetta CLS™ High Content System (PerkinElmer), and images were obtained with Harmony 4.9 software and analyzed with Image J software.
6
Mitochondrial Mass Quantification Protocol
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To determine mitochondrial mass in tissue homogenates, 5 µg of heart or 10 µg of liver in 20 µl of MAS was placed in a clear-bottom black 96-well microplate. Then, 130 µl of a 1:2,000 dilution of the 1 mM MTDR FM stock (MTDR; Thermo Fisher Scientific) was added and incubated for 10 min at 37°C. The plate was centrifuged at 2,000g for 5 min at 4°C (without brake), and the supernatant was carefully removed. Finally, 100 µl of PBS was added per well and MTDR fluorescence measured (λexcitation = 625 nm; λemission = 670 nm). Mitochondrial content was calculated as MTDR signal (minus blank) per microgram of protein. Homogenates and cell lysates were also imaged after the addition of MTDR with a PerkinElmer Operetta CLS high-content system (20× objective). Analysis was performed with PerkinElmer Harmony software (Harmony 4.1) by measuring mean MTDR fluorescence intensity in the whole imaged field.
7
ROS Detection in Cells
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Cells grown on a 96-well CellCarrier (6055300, PerkinElmer, 1 × 104 cells/well) were treated with different media for 24 h. For ROS detection, cells were processed according to the manufacturer’s protocol (ab113851, Abcam). Cells were imaged with a filter set appropriate for fluorescein isothiocyanate (FITC) using a Perkin Elmer Operetta CLS High Content System.
8
Quantifying DNA Damage Response
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Cells (5 × 103/well) were plated into 96-well plates (Cell Carrier Ultra-96; PerkinElmer, Waltham, MA, USA) and cultured overnight. After IR with 5 Gy, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized, and then stained with Alexa Fluor 488-conjugated anti-γ-H2AX Ab and Hoechst 33342. Cell images were analyzed using an Operetta CLS High-Content System (PerkinElmer).
For three-color imaging, cells expressing a GFP-APOBEC3B fusion protein were stained with an anti-γ-H2AX antibody, followed by a secondary antibody conjugated to Alexa Fluor 594, and Hoechst 33342.
For three-color imaging, cells expressing a GFP-APOBEC3B fusion protein were stained with an anti-γ-H2AX antibody, followed by a secondary antibody conjugated to Alexa Fluor 594, and Hoechst 33342.
9
Quantifying E-Cadherin and Vimentin in GFP-Labeled Cells
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HCT116 H2B GFP or HT29 H2B GFP cells were seeded on 24-well cell culture plates and allowed to grow to 70% confluency. Cells were washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, #15710), incubated for 15 minutes, and permeabilized with 1% saponin (Sigma Aldrich, #84510). Blocking buffer of 2% bovine serum albumin (BSA) (Millipore, #260-500GM) and primary antibodies were incubated overnight at 4°C before a 2-hour incubation with secondary antibodies (1:100, Molecular Probes, Invitrogen, #A21428 and #A21235) diluted in blocking buffer. The primary antibodies used for these studies were anti-E-cadherin (1:25; Abcam, #ab1416) and anti-vimentin (1:50; Abcam, #ab92547). Cells were imaged using the Perkin Elmer Operetta CLS High Content System. Quantitation of E-Cadherin+ and Vimentin+ tumor cells (identified by GFP) was performed using the Perkin Elmer Harmony software.
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