Targeting efficiency mediated by Cas9 variants was calculated by monitoring the decrease in tdTomato fluorescence within a single cell. Equal numbers of cells (1.8 × 105 cells/cm2) were plated on Nunc Lab-Tek II two-well chambered coverglass. To visualise individual cells, their nuclear were stained with 10 µg/ml DAPI for 25 min. Blue and red fluorescence images were acquired on an Olympus IX71 inverted fluorescence microscope with a 0.75 NA 40× objective and Orca-Flash4.0 V2 Digital CMOS camera (Hamamatsu). We measured the intensity of tdTomato by averaging the intensity within individual cells using Volocity (Perkin Elmer). The efficiency was determined by pooling the intensity data of individual cells obtained from at least three independent transformations per vector. Cells that exhibited lower red fluorescence than tdTomato knock-in cells were defined as knockouts. Targeting efficiencies of knock-ins were calculated by PCR using primers flanking the target sites; efficiencies of point mutations were calculated using primers with substituted nucleotides at the 3′ ends (Table S4 ).
Orca flash4.0 v2 digital cmos camera
Manufactured by Hamamatsu Photonics
Sourced in Germany, Japan
The ORCA-Flash4.0 V2 Digital CMOS camera is a scientific imaging device designed for high-performance applications. It features a large sensor with a high pixel count, enabling the capture of detailed, high-resolution images. The camera utilizes CMOS technology, providing fast readout speeds and excellent quantum efficiency. The ORCA-Flash4.0 V2 is a versatile tool suitable for a wide range of scientific and research applications.
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Lab products found in correlation
Axio observer, by Zeiss (4 mentions)
Nis element, by Nikon (4 mentions)
Fura red, by Thermo Fisher Scientific (3 mentions)
Sola se 2 365, by Lumencor (3 mentions)
Ff444 521 608 di01 dichroic beamsplitter, by IDEX Corporation (3 mentions)
Nis elements software, by Nikon (3 mentions)
Volocity, by PerkinElmer (2 mentions)
Hxp 120 illuminator, by Zeiss (2 mentions)
Zen blue software, by Hamamatsu Photonics (2 mentions)
Las af software, by Leica (2 mentions)
Af7000, by Leica (2 mentions)
Eclipse ti e, by Nikon (2 mentions)
Nis elements ar microscope imaging software, by Nikon (2 mentions)
Spectra light engine illumination source, by Lumencor (2 mentions)
Furared, by IDEX Corporation (2 mentions)
Las x software, by Leica (2 mentions)
Matlab software, by MathWorks (2 mentions)
Eclipse ti inverted microscope, by Nikon (2 mentions)
Solution and chamber heaters, by Warner Instruments (2 mentions)
Glass bottomed imaging chamber, by Warner Instruments (2 mentions)
42 protocols using orca flash4.0 v2 digital cmos camera
1
Quantifying Cas9 Variant Targeting Efficiency
2
Biofilm Growth Visualization using Fluorescent Microscopy
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To record the biofilm growth, we use Axio zoom technology to make a set of time-lapse widefield images. V16 microscope with a Plan-NeoFluar Z 1.0× objective (NA 0.25) (Zeiss International, Oberkochen, Germany), HXP 200 C metal halide illumination module (Zeiss International, Oberkochen, Germany), and a 16-bit Hamamatsu ORCA-Flash4.0 V2 Digital CMOS camera (Hamamatsu Photonics, Hamamatsu City, Japan) to detect the emitted light. The red mkate2 fluorescent protein was imaged using a Zeiss 63 HE filter set (Ex: BP 572/25; Em: BP 629/62) (Zeiss International), the cyan fluorescent protein was imaged using a Zeiss 47 HE filter set (Ex: BP 436/25; Em: BP 480/40) (Zeiss International), and the yellow citrus fluorescent protein using a Zeiss 46 HE filter set (Ex: BP 500/25; Em: BP 535/30) (Zeiss International).
3
Visualizing Protein Binding with Fluorescence
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The cells were stained with the FITC-labeled EntK1 as described for the binding assay. After discarding the remaining binding buffer, cells were resuspended in 25 μl of PBS, spotted on a microscopy slide, and overlayed with 2% low melting agarose in PBS to immobilize the cells. Phase-contrast images and FITC fluorescence images were obtained using a Zeiss Axio Observer with ZEN Blue software and an ORCA-Flash 4.0 V2 Digital CMOS camera (Hamamatsu Photonics) using a 100 × phase-contrast objective. The excitation light source was an HXP 120 Illuminator (Zeiss).
4
Fluorometric Analysis of Intracellular Calcium
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For the fluorometric analysis we used Fura-2, which is a ratiometric Ca2+ sensitive fluorescent dye. Cells were loaded with 2 μM Fura-2 AM during 30 min on coverslips pretreated with 0.01% Poly-L-Lysine for at least 30 min (to attach the cells to the glass). We measured the time course of [Ca2+]i in cells in PSS and at room temperature in a single cell imaging setup composed of an inverted Zeiss Axio Vert.A1 microscope equipped with a 40X Fluor/1.3 NA objective. Fluorescent illumination was provided by a monochromator (Polychrome V, Till Photonics). The cells were excited alternatively at 340 and 380 nm during 50 ms and the emission light of wavelengths higher than 510 nm was collected. F340/380 ratio was collected every 5 s using an ORCA-Flash4.0 V2 Digital CMOS Camera (Hamamatsu Photonics) operated with the ZEN 2 pro software (Zeiss). At least 200 cells were recorded for each independent preparation. For the thermal stimulation, the cells were perfused with preheated PSS. The temperature solution was regulated with the temperature control system TC-20 (NPI Electronic).
5
Visualizing Rad52 Protein Localization
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In order to visualize Rad52YFP, cells carrying plasmid pWJ1213 were fixed with 2.5% formaldehyde in potassium phosphate at pH 6.4 for 10 min, washed twice with potassium phosphate at pH 6.6, and stored in potassium phosphate at pH 7.4. Cells were permeabilized with 80% ethanol for 10 min and resuspended in 0.5 μg/ml 4′,6‐diamidino‐2‐phenylindole (DAPI). At least 250 cells derived from three independent experiments were analyzed for each time point. Images were acquired with a 63× objective on a wide‐field fluorescence microscope (AF7000, Leica) equipped with an ORCA‐Flash 4.0 V2 digital CMOS camera (Hamamatsu) under the control of LAS AF software (Leica). Images were processed with ImageJ software (https://imagej.nih.gov/ij/ ). To analyze spindle formation, immunofluorescence of tubulin was performed as described (Matos et al, 2013 ), and images were acquired with a 63× objective on an Axio Imager (ZEISS) equipped with a Hamamatsu CCD Camera under the control of Volocity software.
6
Identification and Characterization of Circulating Tumor Cells
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CTCs were assessed by immunofluorescence staining. The following antibodies were used to identify CTCs and assess EPCAM and TROP-2 staining as indicated in the figure legends: Anti-CD45 (BioLegend Cat# 304018, RRID:AB_389336), Anti-CD34 (BioLegend Cat# 343508, RRID:AB_1877133), Anti-CD11B (BioLegend Cat# 101218, RRID:AB_389327), Anti-CD66B (BioLegend Cat# 305109, RRID:AB_2563170), Anti-Pan cytokeratin (BioLegend Cat# 628602, RRID:AB_439775 or Abcam Cat# ab49779, RRID:AB_869395), Anti-Androgen Receptor (Cell Signaling Technology Cat# 5153S, RRID:AB_10692774), Anti-EPCAM (Abcam Cat# ab112068, RRID:AB_10861805) or Anti-TROP-2 (BD Biosciences Cat# 940370, RRID:AB_2876239) and Hoechst 33342 (Thermo Fisher Scientific). Extracellular antibodies were stained at 4°C for 30 minutes. For intracellular and nuclear staining of cells (Fig. 2 ), cells were stained as described by Sperger and colleagues (30 (link)). For intracellular staining (Fig. 4 ), cells were permeabilized, stained, and washed with BD Perm/Wash. Images were taken with a 10x objective using Nikon Eclipse Ti-E with an ORCA-Flash 4.0 V2 Digital CMOS camera (Hamamatsu) and NIS-Elements AR Microscope Imaging Software (RRID:SCR_014329, Nikon Instruments). Images were background subtracted, and CTCs were determined by Hoechst-positive staining, cytokeratin+ and CD45−/CD34−/CD66b−.
7
Single-Molecule FISH for HTLV-1 Transcripts
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HTLV-1-infected T cell clones were subjected to single-molecule RNA-FISH, targeting the plus- or minus-strand transcripts of HTLV-1, following the protocol described previously
17 (link). The coverslips were imaged with an Olympus IX70 inverted widefield microscope with a 100× 1.35NA UPlanApo oil objective lens, a Spectra Light Engine illumination source (Lumencor) and an ORCA-Flash 4.0 V2 digital CMOS camera (Hamamatsu).
17 (link). The coverslips were imaged with an Olympus IX70 inverted widefield microscope with a 100× 1.35NA UPlanApo oil objective lens, a Spectra Light Engine illumination source (Lumencor) and an ORCA-Flash 4.0 V2 digital CMOS camera (Hamamatsu).
8
Fluorescence Microscopy Protocol for High-Resolution Imaging
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Fluorescence
microscopy was carried out using a Zeiss Axio Observer 3 microscope
with a 63× oil immersion objective, NA = 1.4 (Carl Zeiss Microscopy,
LLC., White Plains, NY), a Lumencor Spectra III, LED Light Engine,
and a Hamamatsu ORCA Flash 4.0 V2 Digital CMOS camera (Hamamatsu Photonics
K.K., Hamamatsu City, Japan). The microscope was operated using μmanager
software.23 (link) Additional microscopy settings
are given in theSupporting Information .
microscopy was carried out using a Zeiss Axio Observer 3 microscope
with a 63× oil immersion objective, NA = 1.4 (Carl Zeiss Microscopy,
LLC., White Plains, NY), a Lumencor Spectra III, LED Light Engine,
and a Hamamatsu ORCA Flash 4.0 V2 Digital CMOS camera (Hamamatsu Photonics
K.K., Hamamatsu City, Japan). The microscope was operated using μmanager
software.23 (link) Additional microscopy settings
are given in the
9
Agarose Pad Microscopy Protocol
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One percent agarose in water were made and heated using a microwave until the agarose has completely dissolved. 200 μL of molten agarose was added onto a microscope glass slide and a coverslip placed on top. When the agarose pad has dried and the sample is ready for observation, 2 μL of sample was applied to a prepared agarose pad and a cover slip placed over them. Images were captured on a Leica DMi8 premium-class modular research microscope with a Leica EL6000 external light source (Leica Microsystems), using an ORCA-Flash4.0 V2 Digital CMOS Camera (Hamamatsu) at 100x magnification.
10
Widefield Microscopy Imaging Protocol
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All coverslips were imaged using an Olympus IX70 inverted widefield microscope with a 100x-1.35NA UPlanApo oil objective and a Spectra Light Engine illumination source (Lumencor), with a resolution of 64 nm per pixel, and a spacing of 300 nm per optical slice. Z-offset was applied to correct for chromatic aberration, and exposures were adjusted for each respective wavelength to optimize the signal-noise ratio and minimize photobleaching. Excitation filters used were 390/18 (DAPI), 575/25 (
hbz-Quasar 570) and 632/22 (plus-strand-Quasar 670), with corresponding emission filters of 457/20, 632/60 and 692/40. A Coolsnap HQ camera (Roper Scientific) was used for initial images; the camera was later replaced with an ORCA-Flash 4.0 V2 digital CMOS camera (Hamamatsu).
hbz-Quasar 570) and 632/22 (plus-strand-Quasar 670), with corresponding emission filters of 457/20, 632/60 and 692/40. A Coolsnap HQ camera (Roper Scientific) was used for initial images; the camera was later replaced with an ORCA-Flash 4.0 V2 digital CMOS camera (Hamamatsu).
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