Approximately 0.25 × 106 U2OS.eGFP-PEST cells were nucleofected with 300 ng of Cas9 expression plasmid and 30 ng of eGFP-targeting gRNA expression plasmid using the SE Cell line 4D-Nucleofector X Kit (Lonza) and DN100 pulse program. For anti-CRISPR screening, approximately 0.25 × 106 U2OS.eGFP-PEST cells were nucleofected with 300 ng of Cas9 expression plasmid, 30 ng of eGFP-targeting gRNA expression plasmid, and 50 ng of AcrIIA4 expression or 300 ng of Cas9-P2A-CSD-AcrIIIA4 plasmid and 30 ng eGFP-targeting gRNA expression plasmid using the SE Cell line 4D-Nucleofector X Kit (Lonza) and DN100 pulse program. Approximately 20,000 transfected cells were seeded per well in 100 µL of DMEM medium in a 96-well plate (Perkin Elmer cell carrier) and incubated for 24 h at 37°C with the indicated amount of pomalidomide. Cells were fixed in 4% paraformaldehyde and stained by HCS NuclearMask Blue stain (Invitrogen). Imaging was performed using an Operetta CLS microscope and the Operetta Harmony 4.8 (PerkinElmer) software was used for the analysis.
Operetta cls microscope
Manufactured by PerkinElmer
The Operetta CLS microscope is a high-content screening (HCS) system designed for automated image acquisition and analysis. It features a compact and modular design, offering flexibility in configuration to meet diverse research needs. The Operetta CLS provides high-quality, confocal-like images with advanced analysis capabilities, enabling researchers to gain deeper insights into cellular processes and drug discovery applications.
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2 protocols using operetta cls microscope
1
CRISPR Assay with Anti-CRISPR Screening
2
Efficient Plasmid and RNP Transfection for U2OS Cells
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For plasmid transfection, approximately 20 000 U2OS.eGFP-PEST cells were nucleofected with 300 ng of Cas9 expression plasmid and 30 ng of gRNA expression plasmid using an SE Cell line 4D-Nucleofector X kit (Lonza) following the manufacturer’s protocol. For RNP transfection, wild-type Cas9 (10 pmol of Cas9 and 12 pmol of gRNA) and NL-FKBP-Cas9 (15 pmol of Cas9 and 18 pmol of gRNA) were nucleofected to 2 × 105 U2OS.eGFP.PEST cells. Cells were plated at 14 000 transfected cells per well in 100 μL of DMEM medium in a 96-well plate (Corning) and incubated for 48 h at 37 °C with the indicated amount of compound. Cells were fixed in 4% paraformaldehyde and stained by HCS NuclearMask Blue stain (Invitrogen). Imaging was performed using an IXM ImageXpress microscope or Operetta CLS microscope followed by MetaXpress analysis or Operetta Harmony 4.8 (PerkinElmer) software.
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