Automated confocal fluorescence microscopy using the IN Cell Analyzer 3000™ (GE Healthcare Bio-Sciences, Little Chalfont, UK) has been described in detail [29 ]. For our experiments, we employed the 364 laser line combined with a 450BP25 emission filter for Hoechst 33342™, the 647 nm laser line combined with a 695BP55 emission filter for Alexa Fluor 647. Fluorescence emission was recorded separately in the blue and red green channels, applying flat field correction for inhomogeneous illumination of the scanned area for each of the three channels.
In cell analyzer 3000
Manufactured by GE Healthcare
Sourced in United Kingdom
The IN Cell Analyzer 3000 is a high-content screening system designed for cellular imaging and analysis. It provides automated, quantitative, and high-throughput cellular imaging capabilities for a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
4 protocols using in cell analyzer 3000
1
Automated Confocal Fluorescence Microscopy
2
High-Throughput Imaging Assay Setup
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO); tissue culture reagents and supplies from Life Technologies (Carlsbad, CA), Corning (Tewksbury, MA) or Thermo-Fisher Scientific (Waltham, MA). Draq5 was purchased from Biostatus (Shepshed, UK). Multiwell plates were purchased from Greiner BioOne (Monroe, CA), Corning or Matrical Bioscience (Spokane, WA). Bulk reagents (cells, medium, etc.) were dispensed to all plate wells with a Multidrop Combi (Thermo-Fisher) or a Matrix Wellmate fitted with plate stackers (Thermo-Fisher), and compounds transferred with a CyBi®-Well 384-channel simultaneous pipettor (CyBio, Boston, MA). Serial dilutions of compounds were performed using a BioMek FX® (Beckman Coulter, Brea, CA). Multi-well plates were washed with either a Skatron EMBLA-384 (Molecular Devices, Sunnyvale, CA) or Bio-TekEL405TM (Winooski, VT) platewasher; washers were presterilized with bleach, then ethanol and flushed thoroughly with sterile water before use. The IN Cell Analyzer 3000, a confocal line-scanning instrument, along with cell analysis software modules, was purchased from GE Life Sciences (Piscataway, NJ).
3
High-Content Screening of Kinase siRNA Library
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Screens used the kinase-covering component of the Dharmacon siGENOME SMARTpoolTM library. Library pools were mixed at an equimolar ratio with SMARTpoolTM oligonucleotides targeting TP53 or non-targeting oligonucleotide, then reverse transfected at a combined concentration of 20 nM into HCT116-PSLD, seeded in 96-well plates. Transfected cells were incubated for 24 h prior to treatment with CDK4/6i palbociclib (450 nM) or vehicle for 24 h. Plates were fixed in 4% formaldehyde, then stained with Hoechst 33342 DNA dye and imaged using an INCell Analyzer 3000 (GE Healthcare) or an Opera (Perkin-Elmer) high-content imager platform. Data for a minimum of 2000 cells per condition were collected. Data were processed using CellProfiler open-source image analysis software [51 (link)].
4
Screening miR-21 Inhibitors in HeLaS3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To start the assay, HeLaS3-miR-21 EGFPB cell suspensions were dispensed at 1500 cells per well in 40 µL of complete growth media and incubated for 1 d at 37 °C. For the chemical screen, compounds were preplated in an intermediate 384-well polypropylene plate (Thermo Fisher Scientific) at 100 µM in 10% DMSO (v/v) then diluted in complete growth media; 10 µL was transferred into the assay plates at a final concentration of 10 µM in 1% DMSO (v/v). As an internal reference, each assay plate contained 1% DMSO (v/v) as a negative control in column 13 and an antagomir for miR-21 as positive control in column 14. 8 After 3 d of compound treatment, cells were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (w/v) for 20 min followed by nuclei staining in a solution containing 1 µM of Hoechst and 0.05% Triton X-100 (v/v) for 1 h. Cells were washed with PBS, and assay plates were stored at 4 °C until imaging. Images of cells in assay plates were acquired on the IN Cell Analyzer 3000 (INCA3000, GE Healthcare) for EGFP fluorescence intensity and Hoechst-stained nuclei.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!