Hcs nuclearmask blue stain

The reversal of BRD0539-mediated inhibition of SpCas9 was performed using the eGFP-disruption assay, wherein 2×10⁵ U2OS.eGFP.PEST cells were nucleofected with preformed SpCas9:gRNA complex as previously described. Approximately 22,000 transfected cells/well were plated in four replicates in a 96-well plate (Corning® 3904) along with 15 μM of BRD0539 or DMSO. For AcrIIA4 reversibility experiments, a preformed SpCas9:gRNA (10 pmol) was incubated with AcrIIA4 (5×) for 10 min and then was nucleofected to U2OS.eGFP.PEST cells following the aforementioned protocol. The media was swapped with fresh media containing no BRD0539/AcrIIA4 at the indicated time point (2–24 h), and the cells were allowed to grow until 24 h post-nucleofection. The cells were then fixed using 4% paraformaldehyde and imaged with the HCS NuclearMask™ Blue Stain (Life Technologies) as the nuclear counter-staining agent. Imaging was performed with an ImageXpress Micro automated microscope (Molecular Devices) at 10× magnification under three excitation channels (blue, green, and red) acquiring nine sites per well. Images were analyzed using MetaXpress software and data were plotted using GraphPad Prism 6.

Approximately 8,000 cells/well were seeded in a 96-well plate 24 h before transient transfection with a total of 100 ng of DN66 (mCherry-TAG-GFP reporter) and DN78 (SpCas9 and gRNA) plasmids (1:1) using Lipofectamine 2000 (Life Technologies). Transfected cells were incubated with the indicated amount of small molecule or DMSO for 24 h. Cells were then fixed using 4% paraformaldehyde and imaged with the HCS NuclearMask™ Blue Stain (Life Technologies) as the nuclear counter-staining agent. Imaging was performed with an ImageXpress Micro automated microscope (Molecular Devices) at 4× magnification under three excitation channels (blue, green, and red) with nine acquisition sites per well. Images were analyzed in the MetaXpress software to determine the percent of NHEJ, and the data were plotted using GraphPad Prism 6. The Z’-value was calculated from Equation (1), where σ₁ and σ₂ represent the standard deviations of DN66-transfected wells and (DN66+DN78)-transfected wells, respectively, and μ₁ and μ₂ represent the mean %GFP⁻ cell population for DN66-transfected wells and (DN66+DN78)-transfected wells, respectively.

Approximately 8,000 cells/well were seeded into a 96-well plate 24 h before transient transfection with 100 ng of either CgRNA (Addgene Plasmid #64955) or T1gRNA (Addgene Plasmid #62717) plasmids using Lipofectamine 2000 (Life Technologies). Transfected cells were allowed to grow in the indicated amount of small molecule or DMSO for 24 h. Cells were then fixed using 4% paraformaldehyde and were imaged with the HCS NuclearMask™ Blue Stain (Life Technologies) as the nuclear counter-staining agent. Imaging was performed with an ImageXpress Micro automated microscope (Molecular Devices) at 20× magnification under two excitation channels (blue and red) with nine acquisition sites per well. Images were analyzed using the MetaXpress software to determine the percent of mKate2-positive cells, and data were plotted using GraphPad Prism 6. The Z’-value was calculated from Equation (1) where σ₁ and σ₂ represent the standard deviations of CgRNA-transfected wells and T1gRNA-transfected wells, respectively, and μ₁ and μ₂ represent the mean %RFP⁺ cell population for CgRNA-transfected wells and T1gRNA-transfected wells, respectively.

Approximately 200,000 U2OS.eGFP-PEST cells were nucleofected in duplicate with 500 ng of Cas9 and sgRNA expressing plasmids along with a Td-tomato-encoding plasmid using the SE Cell Line 4D-Nucleofector X Kit (Lonza) according to the manufacturer’s protocol. Approximately 30,000 transfected cells per well in five replicates were plated in a 96-well plate (Corning 3904 clear-bottom) and incubated with the indicated quantities of TMP or 4OHT for 48 h. Cells were fixed using 4% paraformaldehyde, and HCS NuclearMask Blue Stain (Life Technologies) was used as the nuclear counterstaining agent. Imaging was performed with an IXM 137204 ImageXpress Automated High Content Microscope (Molecular Devices) at 4× magnification under three excitation channels (blue, green, and red) with nine acquisition sites per well. Images were analyzed in the MetaXpress software, and data were plotted using GraphPad Prism 6.

The RNP was prepared by incubating SpCas9 (10 pmol) with the eGFP-targeting gRNA (12 pmol) for 10 min at room temperature. AcrIIA4 (50 pmol) was added to the RNP, and the mixture was incubated for 5 min at room temperature. Approximately 2×10⁵ U2OS.eGFP-PEST cells were nucleofected with the RNP or RNP-AcrIIA4 mixture using the SE Cell Line 4D-Nucleofector X Kit (Lonza) according to the manufacturer’s protocol. Approximately 2×10⁴ transfected cells were seeded in a well of a 96-well plate (Corning 3904) and were incubated with the indicated amounts of AcrIIA4 protein for 48 h. The cells were fixed with 4% paraformaldehyde, and the nuclei were stained with HCS NuclearMask Blue Stain (Life Technologies). Imaging was performed using an ImageXpress Micro Automated High Content Microscope (Molecular Devices) at 4× magnification under two excitation channels (blue, green) with nine acquisition sites per well. Images were analyzed using the MetaXpress software.