Cells were treated with doxycycline alone or in combination for 72 hours. Cell proliferation was determined by CellTiter 96R AQueous One Solution Cell Proliferation assay kit (Promega, USA) according to manufacturer’s instructions. Cell apoptosis was determined by labeling apoptotic cells with Annexin V-FITC and 7-AAD (BD Biosciences, USA). The percentage of stained cells was then analyzed using flow cytometry and CXP analysis software (Beckman Coulter, USA).
Annexin 5 fitc and 7 aad
Manufactured by BD
Sourced in United States
Annexin V-FITC and 7-AAD are fluorescent dyes commonly used in flow cytometry applications. Annexin V-FITC binds to phosphatidylserine, which is exposed on the surface of apoptotic cells. 7-AAD is a DNA-binding dye that can distinguish between viable, apoptotic, and necrotic cells. These dyes are used together to identify cell populations undergoing different stages of cell death.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Fc500, by Beckman Coulter (2 mentions)
Facsverse system, by BD (2 mentions)
Anti il 17a pe, by BioLegend (2 mentions)
Anti cd3 fitc, by BioLegend (2 mentions)
Anti tnf α pe, by BD (2 mentions)
Anti cd4 pe cy7, by BioLegend (2 mentions)
Anti cd161 apc, by BioLegend (2 mentions)
Anti tcr vα7.2 brilliant violet 421, by BioLegend (2 mentions)
Anti cd8 perp cy5, by BioLegend (2 mentions)
Anti ccr6 pe cy7, by BioLegend (2 mentions)
Anti granzyme b pe cy7, by BioLegend (2 mentions)
Anti cxcr6 pe, by BioLegend (2 mentions)
Anti perforin pe, by BioLegend (2 mentions)
Anti cd69 pe cy7, by BD (2 mentions)
7 aminoactinomycin d 7 aad, by BD (2 mentions)
Anti ifn γ pe cy7, by BD (2 mentions)
Anti pd 1 pe, by BD (2 mentions)
Binding buffer, by Thermo Fisher Scientific (2 mentions)
25 protocols using annexin 5 fitc and 7 aad
1
Doxycycline's Impact on Cell Proliferation and Apoptosis
2
Synergy and Dose-Response Assays for CLL
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For synergy and dose-response determinations, CLL cells were treated in 96-well plates with 6 increasing concentrations of drug for ~18 or ~72 h. Concentration ranges were selected and optimized to ensure that the exponential phase of the dose-response curve was obtained for the majority of the samples. Ranges were 0–160 µM for BEN, 0–80 µM for IDE and CLB, 0–20 µM for IBR and 0–10 µM for FLU. Synergy experiments with DRB were performed using 3 or 4 increasing doses of drug for ~18 h with the concentration ranges 0–40 µM for BEN and IDE, and 0–20 µM for DRB. The concentration of DMSO was constant between samples. After 18 or 72 h incubation, cell death was determined by flow cytometry by annexin-V-FITC and 7AAD (BD Pharmingen, San Jose, CA, USA) [41 (link)]. Cells were stained with for 15 mins with AV/7AAD and analyzed by flow cytometry using a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA) with a 96-well plate adapter. Cells were considered alive when they were double-negative for AV and 7AAD. When CD19 and CD3 were analyzed in the non-CLL donor PBMCs, anti-CD19-APC or anti-CD3-APC (BD Pharmingen) were added in a triple stain with AV/7AAD. Isotype control antibodies were also run for CD19 (anti-mouse-IgG1κ, BD Pharmingen) and CD3 (anti-mouse-IgG2aκ, BD Pharmingen).
3
Cytotoxicity Assay for PT-112 and Cisplatin
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Cytotoxicity assays were performed as follows: 100 µL aliquots of 3 × 104 cells were seeded per well in a 96-well plate, and 10 µM of PT-112 or cisplatin were added and incubated for 24–72 h at 37 °C. Cell death was analyzed using a FACSCalibur flow cytometer (BD, Biosciences) after incubation with annexin-V-FITC and 7-AAD (BD Biosciences, Madrid, Spain) in annexin binding buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES/NaOH, pH 7.4) for 10 min.
4
Quantifying Apoptosis in HSC and HSPC
5
Endoxifen and VAEM Induce Apoptosis
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Steroid depleted cells (2 × 105/well) were treated in 6-well plates for 5 days with 0, 0.1, 1.0 and 10 μM (E/Z)-endoxifen hydrochloride. Each endoxifen concentration was combined with VAEM at concentrations of 0, 0.1, 1, 10 and 100 μg/mL in the presence or absence of 0.5 μM β-estradiol.
To detect apoptotic cell death, 2 × 105 cells were incubated with Annexin V-FITC and 7-AAD (BD Biosciences Pharmingen™, San Diego, CA, USA) at room temperature in the dark. Samples were analyzed in a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). Annexin V-FITC positive and 7-AAD negative cells were identified as early apoptotic, while late apoptotic/necrotic cells were Annexin V-FITC and 7-AAD double positive. Values were given in percent of total cell number.
To detect apoptotic cell death, 2 × 105 cells were incubated with Annexin V-FITC and 7-AAD (BD Biosciences Pharmingen™, San Diego, CA, USA) at room temperature in the dark. Samples were analyzed in a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). Annexin V-FITC positive and 7-AAD negative cells were identified as early apoptotic, while late apoptotic/necrotic cells were Annexin V-FITC and 7-AAD double positive. Values were given in percent of total cell number.
6
Evaluating EZH2 Inhibitors in CML Progenitor Cells
7
Cell Proliferation and Apoptosis Assay
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Cell were treated with single drugor combinations for 72 hours. Cell proliferation activity was evaluated by using the CellTiter 96 AQueous One Solution Cell Proliferation assay kit (Promega, US) according to manufacturer’s instructions. Assessment of apoptosis was performed by staining cells with Annexin V-FITC and 7-AAD (BD Pharmingen, US) followed by flow cytometry on a Beckman Coulter FC500.
8
Multiparametric Characterization of MAIT Cells
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Flow cytometry was performed as described using the following antibodies. The anti-CD3-FITC, anti-CD161-APC, and anti-TCR Vα7.2-Brilliant Violet 421(Biolegend, USA) were used to identify MAIT cells. Anti-CD4-PE/Cy7, anti-CD8-Perp/Cy5.5, anti-CCR6-PE/Cy7, anti-CXCR6-PE, anti-IL-17A-PE, anti-Granzyme B-PE/Cy7, and anti-Perforin-PE were obtained from Biolegend (USA). Anti-CD69-PE/Cy7, anti-PD-1-PE, anti-IFN-γ-PE/Cy7, anti-TNF-α-PE were obtained from BD (USA). Appropriate isotype control antibodies were used for each staining combination. We used 7-aminoactinomycin D(7-AAD) (BD Biosciences) staining to identify dead cells. Human MR1 tetramers loaded with a potent MAIT cell ligand-5-OP-RU or 6-FP were gifted from professor Li Bai. Apoptosis was allowed to progress and two channels were used to detect annexin V- FITC and 7-AAD (BD Biosciences) to determine the proportions of apoptotic cells. Data acquisition was achieved on BD FACS Verse system (BD Biosciences), and results were analyzed using FlowJo7.6 analysis software.
9
Cell viability and apoptosis assay
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LP-1(wt) and ANBL-6(wt) cells were treated for 4 hours with 1 or 10μM of AT-406 followed by bortezomib treatment (5 or 10nM). After 24 hours the viability was measured with the CellTiter-Glo Luminiscent Viability assay (Promega, Leiden, The Netherlands) according to manufacturer's instructions. The relative amount of viable cells was expressed as percentage of untreated cells.
Apoptosis was measured with Annexin V-FITC and 7-AAD (BD Biosciences) followed by flow cytometric analysis (FACS Canto and Diva software, BD Biosciences) according to manufacturer's instructions.
Apoptosis was measured with Annexin V-FITC and 7-AAD (BD Biosciences) followed by flow cytometric analysis (FACS Canto and Diva software, BD Biosciences) according to manufacturer's instructions.
10
Flow Cytometry Analysis of GFP-Risk1 Constructs
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For flow cytometry analysis, 1 × 106 HeLa cells were transfected with 1 μg of pGFP-vector, pGFP-Risk1 WT, or pGFP-Risk1 H297A plasmid DNA using PolyJet. Twelve hours after transfections, cells were resuspended at a concentration of ∼0.2 × 106 cells/0.1 ml in Cytofix/Cytoperm solution and incubated for 20 min on ice with 20 μl of fluorescein isothiocyanate (FITC)-conjugated anti-active-caspase-3 Ab (BD Pharmingen). Cells were washed 2 times with Perm/Wash buffer and finally resuspended in 200 μl of blocking buffer. Alternatively, transfected cells were stained with annexin V-FITC and 7-AAD using the annexin V-FITC apoptosis detection kit by following the manufacturer’s specifications (BD Pharmingen). For experiments utilizing the PI3K inhibitors, cells were transfected as described above and incubated for 6 h followed by an additional 6 h in the presence of wortmannin (1 to 5 μM), LY294002 (1 to 2 μM), or equal volumes of the diluent DMSO as a control. The percentage of apoptotic cells was assessed by flow cytometry of Risk1+ cells using BD Biosciences FACS Accuri C6 and FCS Express V6 software.
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