Briefly, dissociated PreS1-GFP-HepG2 target cells were mixed with Collagen Type I gel (Rat Tail, Corning) and injected into the dedicated region of the 3D cell-culture chip (DAX-1, AIM Biotech), before gel polymerization, following a previously developed protocol.38 (link),43 ,49 (link) R10 media with 3 μM DRAQ7 (BioLegend) cell-impermeable nuclear dye was then added to the media channels to hydrate the gel, and chips were incubated at 37°C. T cells were stained with 3 μM CellTracker Violet BMQC (Thermo Fisher Scientific) and were injected into one of two media channels flanking the gel region before overnight incubation. 3D confocal images were acquired daily with a high-content imaging system (Phenix, PerkinElmer). T cells from the liquid channel were collected by manual pipetting; then, collagenase solution was injected into the device to retrieve the immune cells migrating in the hydrogel region for flow cytometry analysis on a 4-laser BD LSRII (BD Biosciences).
Draq7
Manufactured by BioLegend
Sourced in United States
DRAQ7 is a fluorescent dye that binds to DNA. It is used for the detection and identification of dead or dying cells in cell biology and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Toluene, by Thermo Fisher Scientific (2 mentions)
Hexane, by Thermo Fisher Scientific (2 mentions)
4 dimethylaminopyridine, by Merck Group (2 mentions)
1 3 dimethylaminopropyl 3 ethylcarbodiimide hydrochloride, by Merck Group (2 mentions)
2 amino 2 hydroxymethyl 1 3 propanediol, by Merck Group (2 mentions)
Chloroform, by Thermo Fisher Scientific (2 mentions)
4 morpholineethanesulfonic acid, by Merck Group (2 mentions)
Anti mouse cd 3 antibody, by BioXCell (2 mentions)
Poly maleic anhydride alt 1 octadecene, by Merck Group (2 mentions)
Lead 2 chloride pbcl2, by Thermo Fisher Scientific (2 mentions)
Sulfur powder sublimed, by Thermo Fisher Scientific (2 mentions)
Di water, by Thermo Fisher Scientific (2 mentions)
Purified anti mouse human pnad antibody, by BioLegend (2 mentions)
Purified anti mouse cd169 antibody, by BioLegend (2 mentions)
Methoxy polyethylene glycol amine mpeg nh2, by Laysan Bio (2 mentions)
Oleic acid, by Merck Group (2 mentions)
Oleylamine, by Merck Group (2 mentions)
1 octadecene, by Merck Group (2 mentions)
Polyacrylic acid, by Merck Group (2 mentions)
25 protocols using draq7
1
3D Co-Culture of HepG2 and T Cells
2
Multiplexed Gene Delivery and Screening
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Firefly luciferase (FLuc) mRNA (5moU) (L-7202), Cre mRNA (5moU) (L-7211) and Cas9 mRNA (5moU) (L-7206) were purchased from TriLink BioTechnologies. DNA barcode (b-DNA) design parameters followed our previous report38 (link). A full list of b-DNA sequences can be found in Supplementary Data 2 . Scrambled negative control sgRNA (Cat#A35526, Thermo Fisher Scientific) and VEGFR2 sgRNA (Synthego) were used as received. All oligonucleotides were purchased from Integrated DNA Technologies and were purified through standard desalting procedures. Luciferase 1000 Assay System (Ref. E4550) and CellTiter-Glo Luminescent Cell Viability Assay Kit (Ref. G7572) were purchased from Promega Corporation. Anti-mouse CD31 antibody (AF488, Cat#102514, Clone#MEC13.3), CD45 antibody (Brilliant Violet 421, Cat#103134, Clone#30-F11), and EpCAM antibody (AF647, Cat#118212, Clone#G8.8) were purchased from BioLegend. Draq7 (Cat#424001, BioLegend) and 7AAD (Cat#A1310, Thermo Fisher Scientific) were used for live/dead staining. Tumor-bearing lung slices were stained with primary antibodies including goat anti-mouse/rat CD31 (Cat#AF3628, R&D Systems) and rabbit anti-mouse GFP (Cat#ab183734, abcam), followed by staining with secondary antibodies including AF488-conjugated donkey anti-rabbit (Cat#A-21206, Thermo Fisher Scientific) and AF555-conjugated donkey anti-goat (Cat#A32816, Thermo Fisher Scientific).
3
FACS Analysis of Spheroid Cultures
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A total of 1 × 105 to 5 × 105 cells were harvested for fluorescence-activated cell sorting (FACS) analysis. To analyze spheroids, a minimum of 240 spheroids were collected and dissociated to single-cell suspension at 37 °C for about 10 min with Accumax (Thermofischer 00-4666-56). Fluorophore-conjugated antibodies and the viability dye were added to the single-cell suspension to perform the immunofluorescence staining.
To differentiate between viable and dead cells for the characterization of the cell populations in the spheroid culture in time, cells were first stained for 15 min with Draq7 (Biolegend, 424001) at room temperature in the dark. Cells were washed twice with PBS before antigen-specific anti-human monoclonal antibodies (Table S4 ) were used to analyze the proteins expressed on the cell surface. Cells were incubated with the antibodies for 45 min on ice in the dark. Bead-based compensation (Beckman Coulter, Nyon, Switzerland, B22804) was performed for each experiment.
Quantification of cell populations was performed after the exclusion of dead cells and focusing on all remaining events. Within this mixture of the different cell types, gates were set based on the selected marker profiles. Gating strategy: all events excluding debris > separation of live and dead cells, excluding dead cells > quantification based on marker profile presented inFigure 1 and Table 1 .
To differentiate between viable and dead cells for the characterization of the cell populations in the spheroid culture in time, cells were first stained for 15 min with Draq7 (Biolegend, 424001) at room temperature in the dark. Cells were washed twice with PBS before antigen-specific anti-human monoclonal antibodies (
Quantification of cell populations was performed after the exclusion of dead cells and focusing on all remaining events. Within this mixture of the different cell types, gates were set based on the selected marker profiles. Gating strategy: all events excluding debris > separation of live and dead cells, excluding dead cells > quantification based on marker profile presented in
4
Lung Epithelial Differentiation from iPSCs/ESCs
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For lung epithelial differentiation, iPSCs/ESCs were MEF-depleted and differentiated according to our previously published serum-free protocol27 (link),38 (link). On day 13, cells were harvested by incubating in 2 mg/ml Dispase (Gibco, #17105-041) for 1.5 h at 37 °C, 2× centrifugation for 2 min at 50 x g, and subsequent single cell dispersion by 0.05% trypsin. Cells were then stained for EPCAM using an anti-CD326 antibody (BD Biosciences, # 563214, 1/500) and the live/dead stain DRAQ7 (Biolegend, #424001, 1/100 dilution). Sorting for EPCAM + , Nkx2-1mCherry+ or GFP+ cells, as indicated in the text, was performed at the BU Flow Cytometry Core Facility using a MoFlo sorter.
5
Apoptosis and Necrosis Detection Assay
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ABT‐737 (APEX Bio), S63845 (ActiveBiochem), TRAIL (Abcam), DRAQ7 (Biolegend), Propidium iodide (PI) (Thermo Fisher Scientific), Digitonin (Sigma Aldrich), CHAPS (Sigma Aldrich) and MitoTracker 561/647 (Thermo Fisher Scientific). ZVAD, NSA, Ferrostatin‐1 from Invitrogen and Cs‐A, MG132, Bortezomib, staurosporine (STS) and rotenone from Sigma Aldrich.
6
Quantifying Organoid Cell Death
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DRAQ7 (Biolegend, 424001), at a final concentration of 5 μM, was added to plates 30 minutes prior to imaging on day 0. Additional 5 μM DRAQ7 was added on day 3 along with fresh medium and drug. Images were acquired with excitation at 633 nm. Areas positive for DRAQ7 were detected within each organoid ROI. ROIs containing one or more areas of DRAQ7 were classified as dead.
7
Biofunctionalized Nanoparticle Synthesis
8
Quantification of Collagen-Induced Cell Death
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Collagen matrices were digested using 2 mg mL−1 type IV collagenase (Thermo Fisher Scientific, Dreieich, Germany) for 15 min under standard cell culture conditions. Afterward, cells were stained with DRAQ7 (dead cell staining dye; Biolegend, San Diego, USA) for 10 min on ice. Cells were analyzed using an Attune NxT Flow Cytometer equipped with an autosampler (Thermo Fisher Scientific, Dreieich, Germany). To analyze cell proliferation, the cell count obtained from the flow cytometer was normalized to the number of cells cultured on a random matrix (0°). The percentage of dead cells was quantified by counting the number of cells that were positive for DRAQ7. At least 20 000 cells were analyzed per condition and replicate. Experiments were performed in six independent replicates.
9
High-Content Screening of Cell Viability
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For HCS experiments, cells were seeded at either 4,000 (HCC4011, H3255) cells per well at least eighteen hours prior to drug treatment on 96-well plates (Corning #3904). For a standard assay setup, one plate per time point was seeded (all at the same time) and the addition of dyes resulted in endpoint analysis. For hypoxia assays, cells were directly placed in a Biospherix C-Chamber set at 0.1% O2 following seeding and subsequent work was done in a hypoxia glove box to maintain constant oxygen control. For glucose modulation, glucose-free media (0 g/L) was supplemented with D-(+)-Glucose solution (Sigma, G8644) to achieve the desired concentration. Prior to imaging, cells were stained with 5 μg/mL Hoechst 33342 (Invitrogen #H21492) and either 5 μg/mL Propidium Iodide (PI) (Invitrogen #P1304MP), 5 μg/mL TO-PRO-3 Iodide (Life Technologies T3605), or 5 μM DRAQ7 (Biolegend, #424001) to identify cells as live or dead, respectively, depending on the fluorescent channels being used for imaging. For morphology assays, cells were stained with 10 μM CellTracker dye (Life Technologies, Orange CMRA #C34551) for thirty minutes and washed with PBS to reduce background.
10
Measuring Cellular Stress and Apoptosis
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Cells were harvested using accutase (#00-4555-56, ThermoFisher scientific), followed by Fc-receptor blocking with anti-CD16/32 (#14-0161-85; ThermoFisher scientific; 1:100, 10 min at 4 °C) prior to staining. Single-cell suspensions were stained with anti-GRP78-A488 (#PA1-014A-A488; ThermoFisher scientific; 1:400, 20 min at 4 °C), APC-Annexin V (#640941; Biolegend, San Diego, CA, USA; 1:20, 15 min at room temperature (RT)), and DRAQ7 (#424001; Biolegend; 1:400, 10 min at RT). Analysis was performed on a GalliosTM Beckman Coulter flow cytometer (Beckman Coulter, CA, USA) with the FlowJo software (FlowJo, LLC, Ashland, OR, USA).
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