Exposed or transfected 661w cells were seeded in 96-well plates (1 × 103 cells / well). At 0h, 24h, 48h and 72h, 10μL Cellell® Counting Kit-8 (CCK-8) solution (Dojindo Laboratories) was added to each well for 1h. The absorbance was measured at 450nm using a microplate reader (Bio-Rad) to detect proliferation. Cells were harvested 72 hours after exposure or transfection, fixed in 75% ethanol, overnight at 4 ° C, and then treated with 20 μl of RNase A at 37 ° C and 400 μl of propidium iodide at 4 ° C for 30 minutes. The cell cycle was detected using BD Accuri C6 flow cytometer (BD Biosciences) and FlowJo 7.6 software analysis. We used two methods to detect apoptosis. One was to collect the cells with trypsin/EDTA (Gibco BRL, USA) and wash them with phosphate-buffered saline (PBS, Gibco BRL). The cells were centrifuged and resuspended in 1mL of buffer and incubated with 5 mU/L of PE and 5 mU/L of 7-AAD for 15 minutes at room temperature (Biovision, CA, USA). The cells were then analyzed immediately using flow cytometry. Analysis of apoptotic cells was carried out using the Caspase-3 spectrophotometric kit (KeyGEN BioTECH, China). Absorbance values were measured on a microplate reader at 400-405 nm to detect cell apoptotic. The experiments were repeated three times.
7 aad
Manufactured by Abcam
Sourced in United States, United Kingdom
7-AAD is a fluorescent dye used for cell viability and DNA content analysis. It binds to DNA and is commonly used to detect late-stage apoptosis and necrosis in cell samples. 7-AAD is impermeable to live cells but can enter cells with compromised membranes, allowing it to distinguish between live, apoptotic, and necrotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 pe, by Abcam (3 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Guava easycyte flow cytometer, by Merck Group (2 mentions)
Rnase a, by Qiagen (2 mentions)
Propidium iodide, by Qiagen (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Facsdiva software 8, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Facsuite software, by BD (1 mentions)
Annexin 5 fitc kit, by Abcam (1 mentions)
Flowjo v 10, by FlowJo (1 mentions)
7 aad, by FlowJo (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
10 protocols using 7 aad
1
Cell Proliferation and Apoptosis Assays
2
Apoptosis and Cell Cycle Analysis
3
Doxycycline-Induced Apoptosis Assay
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22Rv1, DU145, PC3, and VCaP cell lines were treated with 1 µg ml−1 doxycycline for 4 days and stained with annexin V-PE and 7-AAD (Biovision). Apoptosis was then analyzed by flow cytometry according to the manufacturer’s instructions. Live cells (annexin V-negative, 7-AAD-negative), pre-apoptotic (annexin V-positive, 7-AAD-negative) and apoptotic cells (annexin V-positive, 7-AAD-positive) were included in the analysis.
4
Apoptosis and Cell Cycle Analysis
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The HCT116 and SNU283 cell lines were treated with or without α-Amanitin for 2d or doxycycline for 4d at indicated concentrations and stained with annexin V-PE and 7-AAD (Biovision). Apoptosis was analysed by flow cytometry using a Guava EasyCyte Flow Cytometer (Millipore) according to the manufacturer’s protocol. Both pre-apoptotic (annexin V-positive and 7-AAD-negative) and apoptotic (annexin V-positive and 7-AAD-positive) cells were included in the analyses. For cell cycle analysis, cells were fixed in 75% ethanol at −20 °C overnight. The cells were washed with cold PBS, treated with 100 μg of RNase A (Qiagen), and stained with 50 μg of propidium iodide (Roche). Cell cycle profiles were analysed by flow cytometry using the Guava EasyCyte Flow Cytometer (Millipore).
5
Apoptosis Detection by Flow Cytometry
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Apoptotic cells were assayed by flow cytometric analysis of annexin V–allophycocyanine (APC) and 7-amino-actinomycin D (7-AAD) staining. The cells were cultivated in serum-free α-MEM for 24 h to induce apoptosis, collected by trypsin without EDTA (Gibco BRL, USA) and washed with phosphate-buffered saline (PBS, Gibco BRL). After centrifuged and resuspended, the cells were stained with 5 μL Annexin V-APC and 5 μL 7-AAD at room temperature for 15 min (Biovision, CA, USA). Flow cytometry was then used to analyze the cells immediately.
6
Dendritic Cell Nanoparticle Uptake Assay
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Nanoparticle uptake/adherence by the DCs was also evaluated using flow cytometry. DCs were prepared in the same manner as 4.2.11, followed by the addition of FITC-BSA-loaded hybrid cationic CHL NPs (40 µg/40 µL) or FITC-BSA at the same loaded concentration as the cationic CHL NPs (determined in 4.2.4). Media FITC-BSA and FITC-BSA-loaded hybrid cationic CHL NPs were incubated for 1 or 4 h at 37 °C. The un-adhered cells were collected first, followed by the adhered cells, which were treated with TrypLE for 5 min at room temperature. The collected cells were pooled, centrifuged at 200× g for 5 min followed by two washes with PBS. This was followed by resuspension of cells which were incubated in PBS containing 7AAD (abcam, Cambridge, UK) for 10 min, then analyzed using a BD Accuri C6 flow cytometer (BD, Wokingham, UK).
7
Quantifying ICG-001-Induced Apoptosis via FACS
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In order to assess cell apoptosis after treatment with ICG-001, a FACS Annexin V assay was performed. Firstly, 80,000 Cal27 and 150,000 SCC154 cells were seeded into each well on a 12-well plate. On the next day, the cells were either treated with DMSO (vehicle control) or the approximate IC50 concentrations of ICG-001 (5 µM for Cal27 and 1.5 µM for SCC154). After 72 h of treatment, the cells were trypsinized, washed, and pelleted, and the apoptotic cell death was determined by Annexin V (Invitrogen, Thermo Fisher Scientific, R37174, Waltham, MA, USA)/7-AAD (Abcam, ab228563, Cambridge, UK) co-staining, following the instructions from the manufacturer. Data from stained cells were acquired with a BD FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA). Analysis of results was performed with the FACSDiva software 8.0.1 (Becton Dickinson, Franklin Lakes, NJ, USA).
8
Quantifying Apoptosis Induced by BCR-ABL TKIs
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To assess apoptosis induced by BCR-ABL TKIs, HUVECs were stained with Annexin V-FITC (staining of phosphatidylserine) and 7-AAD (DNA staining) in accordance with manufacturer’s instructions (Abcam, Cambridge, UK). Briefly, HUVECs were plated in 12-well plates and exposed to a TKI or 0.2% DMSO (control) for 24 or 72 h. Cells were then stained with Annexin V-FITC and 7-AAD in binding buffer. Stained HUVECs were analyzed on a FACS verse flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). Early apoptotic (i.e., cells with externalized phosphatidylserine but with preserved membrane integrity) and late apoptotic/necrotic cells (i.e., cells with disrupted plasma membrane integrity) were counted based on the relative number of Annexin V-FITC+/7-AAD- and 7-AAD+ cells, respectively, using the BD FACSuite® software.
9
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, scramble siRNA-, and siRNA-transfected cells were washed with PBS and incubated in EtOH 80% for 30 min. After centrifugation and washing steps, cells were resuspended in DAPI/TX-100 solution (9 mL PBS; 1 mL Triton X-100; 10 μL DAPI 1 μg/mL) and incubated for 30 min at room temperature. Cell cycle analysis was next performed by FlowJo v.10.6.1 software, which provides a simple interface to perform sophisticated univariate DNA/Cell Cycle analysis using the Dean-Jett-Fox model71 .
Quantification of apoptotic cells was performed using the Annexin V-FITC Kit (Abcam, cat n° ab14082) and 7-AAD (cat n° 559925), for the exclusion of nonviable cells and detectable in far red, following the manufacturer’s instructions. Briefly, 1 × 105 cells were incubated with 5 µl of both Annexin V and 7-AAD in the proper buffer for 5 min in the dark and then acquired by FACS and analyzed by using FlowJo v.10.6.1.
Quantification of apoptotic cells was performed using the Annexin V-FITC Kit (Abcam, cat n° ab14082) and 7-AAD (cat n° 559925), for the exclusion of nonviable cells and detectable in far red, following the manufacturer’s instructions. Briefly, 1 × 105 cells were incubated with 5 µl of both Annexin V and 7-AAD in the proper buffer for 5 min in the dark and then acquired by FACS and analyzed by using FlowJo v.10.6.1.
10
Isolation and Sorting of Treg and Th17 Cells
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PBMCs were isolated by Ficoll-Hypaque (Lymphoprep, Norway) density gradient centrifugation. The centrifugation was performed at 840 g for 20 min at 20°C. Cells were then cultured in RPMI 1640 medium plus 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 μg/ mL streptomycin for 12 h. Cell-specific antigens were stained for cell sorting, by using PE anti-CD25, APC-Cy7 anti-CD4, APC anti-CD127 antibodies, 7-AAD from Abcam, and APC anti-CCR6 and PE-Cy7 anti-CXCR3 antibodies from Thermo Fisher (Waltham, MA, USA). After the washing step, Treg (CD4+CD25highCD127-) cells and Th17 (CD4+CCR6+CXCR3-) cells were acquired, and dead cells were excluded from analysis by 7-AAD staining. WOLF G2 Cell Sorter and Diva software were used for sorting and analysis.
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