The cultures from the Caov-3 cell lines were washed with PBS. For some experiments, single cells dissociated from tumor spheres were analyzed by the following method. One million trysinized cells were incubated with 7-amino-actinomycin D (BD Biosciences, NJ, USA) only or 7-amino-actinomycin D and a mouse anti-human monoclonal antibody of CD24-FITC (BD Biosciences) with stain buffer (BD Pharmingen™) for 20 min at room temperature in the dark. After washing, the cells were analyzed using a BD FACS Aria™. The fluorescence intensity was analyzed using the BD FACS Diva software program (BD Biosciences).
7 aminoactinomycin d
Manufactured by BD
Sourced in United States, Australia, Canada
7-aminoactinomycin D is a fluorescent nucleic acid-binding dye used in flow cytometry and other bioanalytical applications to detect and quantify DNA content and cell viability.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (14 mentions)
Annexin 5 pe, by BD (13 mentions)
Facsdiva software, by BD (12 mentions)
Annexin 5 fluos, by Roche (12 mentions)
Annexin 5, by BD (11 mentions)
Facscanto 2, by BD (11 mentions)
Facscalibur flow cytometer, by BD (10 mentions)
Muse cell analyzer, by Merck Group (10 mentions)
Lsrii flow cytometer, by BD (8 mentions)
Facscanto 2 flow cytometer, by BD (8 mentions)
Annexin 5 fitc, by BD (7 mentions)
Calcusyn software, by Biosoft (6 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (6 mentions)
Facsaria 2, by BD (5 mentions)
Facscanto, by BD (5 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Golgistop, by BD (4 mentions)
Torin1, by Bio-Techne (4 mentions)
Kaluza analysis 2, by Beckman Coulter (4 mentions)
Ec800, by Sony (4 mentions)
223 protocols using 7 aminoactinomycin d
1
Flow Cytometric Analysis of CD24 in Caov-3 Cells
2
Apoptosis Quantification by Flow Cytometry
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The apoptotic assay was assessed by flow cytometry using Annexin V and 7-aminoactinomycin D (both from BD Biosciences). Cells were seeded in a 25 cm2 flask at a density of 4 × 105 cells and treated with or without EGCG for five days. Then, the cells were harvested and resuspended in 100 µL Annexin V Binding Buffer with 5 µL APC-Annexin V and 5 µL 7-aminoactinomycin D. After co-incubation for 15 min in the dark at room temperature, 100 µL Annexin V Binding Buffer was added and the cells were measured using a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Data acquisition was conducted using BD CellQuest software (version 4.0.2) and analyzed using FlowJo software (version 9.9.5; Tree Star Inc., Ashland, OR, USA).
3
Evaluating Apoptosis in Cell Lines
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MLE-12 cells were stained with PE-Annexin V (BD Pharmingen) and 7-amino-actinomycin D (BD Pharmingen). Splenocytes from PBS- and pristane-injected mice were stained for FITC-conjugated CD4 (BD Pharmingen), PE-Annexin V and 7-amino-actinomycin D. These stained cells were analyzed by flow cytometry. Annexin V-positive and 7-amino-actinomycin D-negative cells were defined as apoptotic cells. The average apoptotic percentages of MLE-12 cells without stimulation, or CD4+ splenocytes from PBS-injected mice on day 14, were determined as 1.0 (apoptotic cell ratios).
4
Antibody-mediated cell cytotoxicity assay
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Cell lines were plated at 2 £ 10 5 cells/well in 96-well plates in complete medium. Antibodies were added at the indicated concentration, followed 5 min later by pooled human serum to a final concentration of 25%. After 4 h of incubation at 37°C, 5 mL 7-aminoactinomycin D (BD Biosciences) was added to each well and the percentage of 7-aminoactinomycin D-positive cells read using a flow cytometer.
5
Immune Cell Staining and Analysis
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The cells were stained with antibodies as previously described (13 (link)), and the following antibodies were used for staining: Annexin V-FITC (556420; BD Biosciences, San Jose, CA, USA) at 1:20, 7-Aminoactinomycin D (559925; BD Biosciences) at 1:20, and anti-human CD274 (PD-L1) PE (12-5983-42; eBioscience, Santa Clara, CA, USA) at 1:20. An isotype-matched immunoglobulin served as a negative control, and dead and/or apoptotic cells were excluded using Annexin V and 7-Aminoactinomycin D. Staining was detected using an LSRII flow cytometer (BD Biosciences).
6
Chondrocyte Proliferation and Apoptosis Analysis
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The mutant or control chondrocytes were plated in six-well plates with the density of 1×105, and cultured in medium with 1% serum for 24 h and then were restimulated with insulin (10 nmol⋅L−1) for an additional 24 h before harvest. For proliferation analysis, BrdU was added to the medium 12 h before harvesting the cells. The cells were then stained for anti-BrdU-APC and 7-amino-actinomycin D following the manufacturer’s instructions (BD Biosciences). For the apoptosis assay, annexin V-PE (BD Biosciences) and 7-amino-actinomycin D were used for staining. Cells were analyzed by FACSCalibur (BD Biosciences) and 20 000 events were collected. The results were analyzed with ModFit LT 3.3 software.
7
Fluorescence Analysis of Cell Surface Markers
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The cultures from the Caov-3 cells were washed with PBS. For some experiments, single cells dissociated from tumor spheres were analyzed by the following method. One million trysinized cells were incubated with 7-amino-actinomycin D (BD Biosciences) only, or with 7-amino-actinomycin D and a mouse anti-human monoclonal antibody of CD24-FITC (BD Biosciences) with a stain buffer (BD Pharmingen™) for 20 min at room temperature in the dark. After washing, the cells were analyzed using a BD FACS Aria™. The fluorescence intensity was then analyzed using the BD FACS Diva software program (BD Biosciences).
8
Multicolor Flow Cytometry Characterization of Immune Cell Subsets
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Cells were labeled with fluorochrome conjugated monoclonal antibodies against the following cell markers: CD11c, MHC class II (MHC-II), CD11b, Ly6C, Ly6G, Siglec-F, CD3, CD4, CD8 and CD44 (BD Pharmigen). 7-Aminoactinomycin D (BD Pharmigen) was used for dead cell exclusion. Cells were labeled with various combinations of mAbs. Dendritic cells were characterized as CD11c+ low autofluorescent, MHCII+, CD11b+ cells [26 (link),27 (link)]. Neutrophils were characterized as CD11c−CD11b+Ly6C+Ly6G+ cells. Eosinophils were characterised as CD11c−CD11b+Siglec F+ cells. T-cells are identified as CD3+ cells with low side scatter. They are further gated as either CD4high or CD8high [4 ]. Data acquisition was performed on a FACSCalibur flow cytometer running CellQuest software. FlowJo software (Tree Star) was used for data analysis.
9
Apoptosis/Necrosis Assay for β-carbolines
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The apoptosis/necrosis assay was performed by double staining with CellEvent Caspase 3/7-FITC (Thermo Fisher Scientific) and 7-aminoactinomycin D (BD Pharmigen) fluorescent dyes. Briefly, the cells (4 × 105) were seeded onto six-well plates and incubated until 70–80% confluency. On the next day, the cells were treated with β-carbolines in a final concentration of 5, 10, and 25 µM. Afterward, the cells were detached with trypsin (Thermo Fisher Scientific), washed twice with Dulbecco’s Phosphate Buffered Saline (Thermo Fisher Scientific), and stained with fluorescent dyes for 30 min at 37 °C in the dark, in line with the manufacturer’s protocol. Cells were analyzed immediately with 488 nm excitation using an Accuri C6 flow cytometer (Becton Dickinson).
10
Flow Cytometric Analysis of Immune Cells
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Flow cytometric analysis of BAL cells was performed to enumerate dendritic cells (DCs), macrophages, neutrophils and CD4+ and CD8+ T cells. BAL cells were labeled with fluorochrome-conjugated monoclonal antibodies against the following cell markers: CD11c, MHC class II (MHC-II), CD11b, Ly6C, Ly6G, CD3, CD4 and CD8 (BD Pharmigen, San Jose, CA, USA). 7-aminoactinomycin D (BD Pharmigen, San Jose, CA, USA) was used for dead cell exclusion. Data acquisition was performed on a FACSCalibur flow cytometer running Cell Quest software. FlowJo software (Tree star Inc., Ashland, Oregon, USA) was used for data analysis. TNF-α protein level in BAL fluid was determined using a commercially available ELISA kit (eBioScience, Vienna, Austria).
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