The detection of apoptosis was carried out by concurrent staining with annexin V–FITC (Thermo Fisher Scientific, Walthan, MA, USA) and 7-aminoactinomycin D (7-AAD) (Beckman Coulter, Marseille, France) following treatment of cell lines and oral keratinocytes by QMTs. Cisplatin was used as a positive control due to its known proapoptotic effects. The Fluorescence Minus One Control (FMO) was used to correct spectral overlap and as a staining control. At least 5000 events were acquired for each condition using the flow cytometer FACSARIA II (BD Biosciences) and FlowJo software (TreeStar, Ashland, OR, USA) was used for data analyses. Early apoptosis was identified in the cells stained only with Annexin-V, late apoptosis or necrosis was identified in the cells stained with both Annexin-V and 7-AAD and unlabeled cells were considered viable.
7 aminoactinomycin d 7 aad
Manufactured by Beckman Coulter
Sourced in United States, France
7-aminoactinomycin D (7-AAD) is a nucleic acid-binding dye used for flow cytometry applications. It is a fluorescent stain that binds to DNA and can be used to distinguish between viable and non-viable cells.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc, by Thermo Fisher Scientific (2 mentions)
Cellquest pro software, by BD (2 mentions)
Fc500 flow cytometer, by Beckman Coulter (2 mentions)
Kaluza flow analysis software, by Beckman Coulter (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Anti ki67 alexa fluor 488 monoclonal antibody, by BD (2 mentions)
Fitc annexinv, by Beckman Coulter (2 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Annexin 5, by Beckman Coulter (1 mentions)
Bromodeoxyuridine (brdu), by BD (1 mentions)
Alexa fluor 488 conjugated anti brdu antibody, by BioLegend (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Ibrutinib pci 32765, by Selleck Chemicals (1 mentions)
Annexin 5 fitc, by Beckman Coulter (1 mentions)
Cellquest, by BD (1 mentions)
Accutase, by Merck Group (1 mentions)
Ow cytometer, by BD (1 mentions)
Dihydroethidium dhe, by Cayman Chemical (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Anti cd8 fitc, by Beckman Coulter (1 mentions)
20 protocols using 7 aminoactinomycin d 7 aad
1
Apoptosis Detection in Cell Lines
2
Cell Cycle and Apoptosis Assay
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1.5 × 105 cells were incubated overnight in a 6-well culture plate and then incubated in fresh medium with 50–200 nM panobinostat and/or 5–20 mM metformin. After 48-hour treatment, they were washed with phosphate-buffered saline (PBS) and harvested by trypsinization. For cell cycle analysis, cells were resuspended in citrate buffer and stained with propidium iodide. For apoptotic cell analysis, cells were stained with annexin V and 7-aminoactinomycin D (7-AAD) according to the manufacturer's protocol (Beckman Coulter, Marseille, France). They were analyzed by flow cytometry using the CellQuest Pro software (BD Biosciences, San Jose, CA, USA). The experiment was performed three times.
3
Pulse-Chase Assay for Cell Proliferation
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For the pulse-chase assay, cells were treated simultaneously with 20 μM BrdU (BD Biosciences) and 25 ng/ml illudin S for 1 h, and then incubated for the indicated periods without the drugs. For pulse-labeling assays, cells were treated with 25 ng/ml illudin S for 1 h, incubated without the drug for the indicated periods, and then treated with 20 μM BrdU for 1 h. Cells were harvested and fixed with 70% ethanol at −20°C. The fixed cells were treated with 2 N HCl supplemented with 0.5% Triton X-100. After washing twice with 0.1 M Na2B4O7 and once with dilution buffer (1% BSA and 0.1% Tween-20 in PBS), the cells were incubated with Alexa Fluor 488–conjugated anti-BrdU antibody (BioLegend). After treatment with 0.2 mg/ml RNase A, DNA was stained with 7-amino-actinomycin D (7-AAD) (Beckman Coulter). Data were collected on an FC500 flow cytometer (Beckman Coulter) and analyzed using FlowJo software (BD Biosciences). Proportions of BrdU-positive cells were calculated by gating using FlowJo software.
4
Murine Antibodies for Lymphocyte Profiling
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Murine monoclonal antibody against CCR7 and CCRL2 and PE-labelled antibodies against CXCR5 and CXCR3 were purchased from R&D Systems. PE-labelled antibodies against CXCR4, PE and FITC-labelled antibodies against CD19, and FITC and PE-labelled antibodies against IgD were purchased from BD. PE-labelled antibodies against IgM were purchased from Biolegend and Beckman Coulter. PE-labelled rabbit α-mouse IgG was obtained from Dako Cytomation. Goat F(ab’)2 anti-human IgM (α-IgM) and IgD (α-IgD) for stimulations and F(ab’)2 of irrelevant specificity as a control were purchased from Southern Biotech. Chemokines were purchased from Peprotech and R&D. Ibrutinib (PCI-32765) was bought from Selleckchem, Bafetinib (INNO 406) from Adooq, and R406 from Riegel Pharmaceuticals. Annexin V-FITC and 7-aminoactinomycin D (7AAD) for determination of cell viability were from Beckman Coulter.
5
Apoptosis Induction Assay Protocol
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Cells were plated for 24 h before induction of apoptosis. After treatment with or without 5-FU, CPT-11, CDDP, or MMC (10 μg/mL each), cells were detached with Accutase (Sigma-Aldrich, St. Louis, MO, USA) and double-stained with annexin V–FITC (eBioscience, San Diego, CA, USA) and 7-amino-actinomycin D (7-AAD; Beckman Coulter, Brea, CA, USA), according to the manufacturer's protocol. Samples were immediately analyzed by FACSCalibur using CellQuest (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (Tree Star, Ashland, OR, USA). The apoptosis proportion was defined as the percentage of the annexin V–FITC-positive cells among total cells.
6
Annexin-V, Cell Cycle, and ROS Analysis
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Flow cytometry was used for analysis of annexin-V assay and cell cycle and evaluation of cellular reactive oxygen species (ROS) production. Brie y, 1.0 10 5 cells were seeded into each well of a 12-well culture plate one day before being cultured for 48 hours under the indicated conditions. Cells were then washed with phosphate-buffered saline and harvested by trypsinization. For annexin-V assay, cells were subjected to annexin V and 7-amino-actinomycin D (7-AAD) double staining following the protocol of the assay kit's manufacturer (Beckman Coulter, Marseille, France). For cell cycle analysis, cells were resuspended in citrate buffer and stained with propidium iodide. For evaluation of cellular ROS production, cells were stained with dihydroethidium (DHE) (Cayman Chemical) according to the manufacturer's protocol. The cells were then analyzed using a ow cytometer and CellQuest Pro Software (BD Biosciences, San Jose, CA, USA). Three independent tests were performed.
7
Comprehensive Lymphocyte Profiling by Flow Cytometry
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Flow cytometric analysis was used to characterize lymphocyte subsets in isolated PBMCs. In brief, frozen PBMC aliquots were quickly thawed and counted with a Neubauer chamber. Discrimination between live and dead cells was carried out prior to analysis with 7-aminoactinomycin D (7-AAD; Beckman Coulter). The following conjugated human antibodies were used: PerCP-anti-CD3 and PCy5-anti-CD3 as pan T-cell marker (Beckman Coulter, CA, USA), FITC-anti-CD4 for T-helper cells, FITC-anti-CD8 for cytotoxic T cells, APC-anti-CXCR3 and PE-anti-CCR4 for Th1 and Th2 cells, PE-anti-FOXP3 for Tregs, and APC-anti-CD19 for B cells. For porcine samples, the following antibodies were used: FITC-anti-CD1 for B cells and APC-anti-FOPX3 for Tregs (Beckton Dickinson, NJ, USA). Lymphocyte apoptosis was analyzed in fresh blood samples by dual selectivity with Annexin V and 7-AAD. In brief, the lymphocyte population was gated, and apoptosis was determined as the percentage of cells positive for Annexin V but negative for 7-ADD.
Samples were analyzed using a BD FACSVerse flow cytometer (standard 2-laser configuration, BD, USA), and a minimum of 10,000 events was acquired. FlowJo 8.7 software (TriStar, Oregon, USA) was used for the analysis of all the acquired data.
Samples were analyzed using a BD FACSVerse flow cytometer (standard 2-laser configuration, BD, USA), and a minimum of 10,000 events was acquired. FlowJo 8.7 software (TriStar, Oregon, USA) was used for the analysis of all the acquired data.
8
Evaluating DC-SIGN Expression and Polyman26 Toxicity
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DC-SIGN expression by iMDDCs was checked by staining with the anti-human DC-SIGN PE antibody (clone AZND1, Beckman Coulter, Milano, Italy). To evaluate Polyman26 toxicity, iMMDCs were incubated with increasing concentration of Polyman26 for 3, 24, and 48 hours, and then labeled with 7-aminoactinomycin D (7-AAD) (Beckman Coulter). Samples were acquired by using a Gallios™ Flow Cytometer and data were analysed with Kaluza® Flow Analysis Software (both Beckman Coulter).
9
Evaluating Immunotoxin Cytotoxicity and Apoptosis
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For evaluation of cytotoxic effects of the immunotoxin, cells were seeded at 2 × 104 cells per 200 μl in 96-well plates. HM1.24-ETA′ or a control toxin was added at the indicated concentrations. After 3 days, vital cell mass was measured using colorimetric tetrazolium (MTT/MTS)-based assays (Cell Proliferation Kit I; Roche, Mannheim, Germany; Promega, Madison, WI, USA). For the detection of early stages of apoptosis and cell death, cells were seeded at 2 × 105 cells per ml in 24-well plates with increasing immunotoxin concentrations. For analyzing the kinetics of apoptosis induction, the immunotoxin was used at 100 ng/ml. Cells were stained with FITC-conjugated annexin V and 7-aminoactinomycin D (7-AAD; Beckman Coulter, Fullerton, CA, USA) according to the manufacturer's protocol, and subsequently analyzed by flow cytometry. For blocking experiments, a 50-fold molar excess of parental antibody was added 30 min before adding immunotoxin.
10
Annexin V-FITC and 7-AAD Apoptosis Assay
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Ice-cold PBS was used to wash the detached cells twice, followed by suspension in 1 ml of Dulbecco’s phosphate-buffered saline (DPBS) and dropwise addition of 4% paraformaldehyde (PFA). Cells were then stained with Annexin V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD) (Beckman Coulter, Brea, CA, USA), and with propidium iodide (PI)/RNase staining buffer, and incubated at room temperature for 30 min in the dark.
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