After a 30 min incubation with different concentrations of H2O2, cells were labeled with phycoerythrin- (PE-) conjugated annexin V and 7-amino-actinomycin (7-AAD) (BD Pharmingen, USA) following the kit instructions. Approximately 5,000 cells were counted from each sample using flow cytometry (BD Biosciences, USA), and early apoptotic cells were defined as annexin V positive/7-AAD negative.
7 amino actinomycin 7 aad
Manufactured by BD
Sourced in United States, Belgium
7-amino-actinomycin (7-AAD) is a fluorescent dye used in flow cytometry applications. It binds to DNA and emits fluorescence, allowing the identification and quantification of cells at different stages of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (5 mentions)
Annexin 5, by BD (5 mentions)
Accuri c6, by BD (5 mentions)
7 aad, by BD (4 mentions)
Annexin 5 apc, by BD (3 mentions)
Annexin 5 phycoerythrin pe, by BD (3 mentions)
Annexin 5 pe, by BD (3 mentions)
Winmdi 2, by BD (3 mentions)
Aldefluor kit, by STEMCELL (2 mentions)
Version 7, by FlowJo (2 mentions)
Pkh26, by Merck Group (2 mentions)
Facscanto 2, by BD (2 mentions)
Propidium iodide, by BD (2 mentions)
Annexin 5 phycoerythrin, by BD (2 mentions)
Annexin 5 binding buffer, by BD (2 mentions)
Baby rabbit complement, by Bio-Rad (2 mentions)
Facscan, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Facsaria 3 flow cytometer, by BD (2 mentions)
Annexin 5 allophycocyanin apc, by BD (2 mentions)
65 protocols using 7 amino actinomycin 7 aad
1
Annexin V and 7-AAD Apoptosis Assay
2
Quantifying Apoptosis in Liver Tissue
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Apoptosis was determined by dual staining with APC Annexin V (BD Biosciences) and 7-amino-actinomycin (7-AAD) (BD Biosciences) with subsequent flow cytometry analysis. The relative proportion of Annexin V-positive and 7-AAD negative cells was determined using the ModFitLT software (Becton Dickinson, San Diego, CA) and counted as early apoptotic cells (Annexin V-positive, 7-AAD-negative), late apoptotic cells (Annexin V-positive, 7-AAD-positive), necrotic cells (Annexin V-negative, 7-AAD-positive) and viables (Annexin V-negative, 7-AAD-negative),.
In addition, terminal deoxynucleotidyl TUNEL staining was also employed for detection of apoptosis in liver sections (Promega, Madison, WI). The apoptosis index was calculated as the percentage of TUNEL-positive cells which showed clear brown nuclear staining (n >1000).
In addition, terminal deoxynucleotidyl TUNEL staining was also employed for detection of apoptosis in liver sections (Promega, Madison, WI). The apoptosis index was calculated as the percentage of TUNEL-positive cells which showed clear brown nuclear staining (n >1000).
3
Isolation and Characterization of Stem-like Cancer Cells
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Cells were filtered using 5 ml polystyrene round-bottom tube with cell strainer caps (BD Biosciences; Bedford, MA, USA). 7-aminoactinomycin (7-AAD; BD Biosciences) was used to eliminate dead cells. ALDH activity was quantified using the Aldefluor kit (StemCell Technologies; Cambridge, MA, USA). Single cell suspensions of 2 × 106 cells/ml were prepared and incubated with 5 μl Aldefluor substrate, or 5 μl diethylaminobenzaldehyde (DEAB, ALDH activity inhibitor) for 40 min at 37 °C. Then, cells were washed and exposed to 1:100 anti-CD44 (APC; BD Biosciences) for 30 min at 4 °C. When required, cells were sorted according to ALDH activity and expression of CD44. Alternatively, cells were stained with combination of AldeRed 588-A (Cayman; Ann Arbor, MI, USA) and CD44 violet (V450, BD Biosciences) for experiments with GFP-transfected cells. For apoptosis studies, cells were exposed to rapamycin, temsirolimus, cisplatin (Teva; Cincinnati, OH, USA) or paclitaxel (Fresenius Kabi; Grand Island, NY, USA) for 24 h. Cells were incubated with Aldefluor substrate (StemCell Technologies), CD44 violet (V450, BD Biosciences), and 1:100 Annexin V (APC; BD Biosciences) for 20 min at room temperature. Cells treated with DMSO were used as control for the Annexin V studies.
4
Apoptosis Quantification by Flow Cytometry
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After drug treatment, cells were double-stained with Annexin V-phycoerythrin (PE) and 7-amino-actinomycin (7-AAD) (BD Biosciences) at room temperature in the dark as described in the vendor’s protocol, followed by flow cytometry analysis within 1 h (BD Biosciences). We analyzed proportions of apoptotic cells using FlowJo Version 7.6.1 software (FlowJo, Ashland, OR).
5
Nutlin-3a Induced Apoptosis in SiHa and HEK Cells
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SiHa cells or HEK cells, cultured in 96-well plates at a concentration of 4.0 × 103/well or 8.0 × 103/well, were transduced or transfected following the above steps. Then they were treated with or without 40 µg ml−1 or 20 µg ml−1 of nutlin-3a for one to five days. Cellular viability assay was detected using water-soluble tetrazolium 1 (WST-1) assay at each time point according to the protocol. Each treatment was performed in quadruplicate, and the experiment was repeated three times.
At 72 hpt, the cells in 6-well plates were harvested and resuspended in 100 µl of 1 × Annexin-V binding buffer. The supernatant was incubated with 5 µl of phycoerythrin (PE)-conjugated Annexin V (BD Pharmingen, USA) and 5 µl of 7-amino-actinomycin (7-AAD, BD Pharmingen) for 15 min at room temperature in the dark, and then 400 µl of 1 × buffer was added to each tube. Flow cytometric analysis (BD FACSCalibur, BD Biosciences, USA) was performed within 1 h of staining. Each experiment was performed with at least three biological replicates.
At 72 hpt, the cells in 6-well plates were harvested and resuspended in 100 µl of 1 × Annexin-V binding buffer. The supernatant was incubated with 5 µl of phycoerythrin (PE)-conjugated Annexin V (BD Pharmingen, USA) and 5 µl of 7-amino-actinomycin (7-AAD, BD Pharmingen) for 15 min at room temperature in the dark, and then 400 µl of 1 × buffer was added to each tube. Flow cytometric analysis (BD FACSCalibur, BD Biosciences, USA) was performed within 1 h of staining. Each experiment was performed with at least three biological replicates.
6
Leukemic Cell Viability Assay
7
Apoptosis and Necrosis Quantification
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AR42J cells (1×105) were collected to detect apoptotic and necrotic indices using the APC Annexin V Kit (BD Biosciences) in conjunction with the vital dye 7-amino-actinomycin (7-AAD) (BD Biosciences). Briefly, 1×105 of AR42J cells were trypsinized, washed twice with clod PBS, then resuspended in 500 μl 1x binding buffer. Samples received 5 μl each of APC Annexin V and of 7-AAD, were gently vortexed, and incubated for 15 min at 25°C in the dark. Each sample was subsequently analyzed in triplicate by using flow cytometry (BD Biosciences).
8
Fluorescent Apoptosis and Necrosis Assay
9
NK Cell-Mediated Cytotoxicity Assay
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NK cytotoxic activity was conducted as previously described [52 (link)]. NK cells were labelled with Paul Karl Horan (PKH-26) (3.5 μl/test) (2 × 10− 6 M) for 5 min (Sigma-Aldrich, St. Louis. MO, USA) and incubated with K562 cells for 4 h at 37 °C with 5% CO2 in RPMI-1640 supplemented with 10% FBS. The concentration of NK cells and K562 cells were adjusted to 5 × 105 cells/ml and 1 × 106 cells/ml, respectively. During incubation, cells were combined at E:T ratios of 12.5:1 and 6.25:1 in addition to controlling samples. Control and RTX treated cells were stained using Annexin V (2.5 μl/test) (BD Bioscience, San Jose, CA, USA) and 7-amino-actinomycin (7-AAD) (2.5 μl/test) (BD Bioscience, San Jose, CA, USA) to determine apoptosis using flow cytometry analysis recording 10,000 events (Additional file 1 ). E:T ratio of 12.5: 1, 6.25 and control were used to assess cytotoxic activity [38 (link), 40 , 53 (link)]. NK cytotoxic activity was calculated as percent specific death of the K562 cells for the two E:T ratios as previously described [40 , 52 (link)]. The percentage of target cell lysis was calculated from:
10
Apoptosis Quantification by Flow Cytometry
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For the apoptosis assay, single cell suspensions were stained with APC-Annexin V (Biolegend, San Diego, CA, USA) and 7-Amino-Actinomycin (7-AAD) (BD Pharmingen, San Jose, CA, USA) in an Annexin V binding buffer according to the manufacturer’s instruction. Cells were analyzed using a BD FACS CantoII. FACS data was quantified using the FlowJo software (Tree Star, Ashland, OR, USA).
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