Apoptosis was analyzed using an ApoScreen Annexin V Apoptosis Kit (SouthernBiotech, Birmingham, AL, USA). Briefly, 1 × 106 cells were washed twice with cold PBS and stained with annexin V-FITC and propidium iodide (PI). The stained cells were analyzed using a BD FACSCanto II (BD Biosciences) running BD FACSDiva software (BD Biosciences). Data were analyzed using FlowJo version 10 (Tree Star Inc.).
Aposcreen annexin 5 apoptosis kit
Manufactured by Southern Biotech
Sourced in United States
The ApoScreen Annexin V Apoptosis Kit is a laboratory equipment product designed to detect and quantify apoptosis, a type of programmed cell death. The kit utilizes annexin V, a calcium-dependent phospholipid-binding protein, to identify cells undergoing apoptosis. This product provides the necessary reagents and protocols to perform the annexin V-based apoptosis assay.
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22 protocols using aposcreen annexin 5 apoptosis kit
1
Apoptosis Analysis via Annexin V-FITC
2
Apoptosis Analysis by Cleaved PARP
3
Profiling Tumor Cell Proliferation and Apoptosis
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Following approval by the IRB, PTCL cells were isolated from PBMC of three patients with high circulating tumor burden using standard Ficoll–Hypaque technique (>95% CD3+/CD5– tumor cells). SR, Jurkat and SUP-T1 (T-lymphoblast) cell lines and Sezary HH and HuT-78 cells were obtained from the American Type Culture Collection (ATCC). All cells were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum.
To measure cell proliferation, cells were plated in 96-well plates at 3000/well in 100 μL (6 per sample) and incubated for 72 hours. Viable cells were measured using a CellTiter Aqueous One Solution Cell Proliferation Assay (Promega).
Cell apoptosis was measured in duplicates using the ApoScreen Annexin V Apoptosis Kit as previously described (Southern Biotech) [8 (link)]. For cell cycle analysis, 2 × 105 cells were fixed in ice cold 70% ethanol while being vortexed, incubated on ice for 15 minutes, washed in PBS and resuspended in 250 μl of staining solution containing 20 ng/ml propidium iodide, 200 ng/ml RNAse A (Sigma Aldrich), 0.1% Triton-X 100 and 1 μl CD19-FITC mAb in PBS. Cells were incubated for 15 minutes and submitted to flow cytometry. Analysis was performed using FlowJo software (Tree Star).
Pevonedistat was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited.
To measure cell proliferation, cells were plated in 96-well plates at 3000/well in 100 μL (6 per sample) and incubated for 72 hours. Viable cells were measured using a CellTiter Aqueous One Solution Cell Proliferation Assay (Promega).
Cell apoptosis was measured in duplicates using the ApoScreen Annexin V Apoptosis Kit as previously described (Southern Biotech) [8 (link)]. For cell cycle analysis, 2 × 105 cells were fixed in ice cold 70% ethanol while being vortexed, incubated on ice for 15 minutes, washed in PBS and resuspended in 250 μl of staining solution containing 20 ng/ml propidium iodide, 200 ng/ml RNAse A (Sigma Aldrich), 0.1% Triton-X 100 and 1 μl CD19-FITC mAb in PBS. Cells were incubated for 15 minutes and submitted to flow cytometry. Analysis was performed using FlowJo software (Tree Star).
Pevonedistat was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited.
4
Analysis of Apoptosis and Proliferation
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Cells were transfected with siRNAs and incubated for 24, 48, or 72 h, after which they were treated using a Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific) or ApoScreen Annexin V Apoptosis Kit (Southern Biotech, Birmingham, AL, USA) according to the manufacturers’ instructions. Flow cytometric analyses were performed using a BD FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA) with BD FACSDiva software (BD Biosciences). Data analysis was performed using FlowJo software (FlowJo, LLC, Ashland, OR, USA). Apoptosis was also assessed by detecting caspase-3 activity using an APOPCYTO Caspase-3 Fluorometric Assay Kit (MBL, Nagoya, Japan) and an Infinite M1000 PRO (TECAN, Männedorf, Switzerland) according to manufactures’ instructions.
5
Annexin-V/PI Apoptosis Assay
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Apoptosis was evaluated by Annexin-V/PI staining and flow cytometric analysis using an ApoScreen Annexin V Apoptosis Kit (Southern Biotech, Birmingham, AL, USA) according to the manufacturer’s instructions. Data were analyzed using FlowJo software version 10.
6
Apoptosis and Necrosis Evaluation of Tenocytes
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Semi-confluent human and rat tenocytes from 10 donors were exposed to 0.1% lidocaine, 0.1 U/mL NTP, 0.1% lidocaine with 0.1 U/mL NTP, or a control medium in T-75 flasks for 24 hours. The tenocytes were trypsinized, collected by centrifugation, washed with phosphate-buffered saline, and labelled with annexin V-FITC and propidium iodide (PI) for 15 min according to the manufacturer’s protocol (ApoScreen Annexin V Apoptosis Kit; Southern Biotech, Birmingham, AL, USA). The cells were analyzed using a FACSDiva flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) to count apoptotic (annexin V-positive and PI-negative), necrotic (PI-positive), and viable (annexin V- and PI-negative) cells.
7
Apoptosis and Proliferation Assay
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Cells were transfected with 2.5 μg of a ZNF582-AS1 expression vector or an empty vector as described above. Forty-eight to 96 h after transfection, the cells were harvested and subjected to flow cytometry analyses using ApoScreen Annexin V Apoptosis Kit (Southern Biotech) or Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Life Technologies) and BD FACSCanto II (BD Biosciences). Data analysis was performed using the FlowJo software (FlowJo, LLC).
8
Cell Cycle and Apoptosis Analysis
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Cell cycle analysis was performed with flow cytometry using a propidium iodide cell cycle detection kit (Beyotime, Beijing, China) and a flow cytometer (BD, Franklin Lakes, NJ, USA). Apoptosis was analyzed using the ApoScreen Annexin V Apoptosis Kit (SouthernBiotech, Birmingham, AL, USA) according to the manufacturer's instructions.
9
Annexin-V Apoptosis Assay Protocol
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An ApoScreen Annexin-V apoptosis kit (Southern Biotech) was used for detecting apoptosis and necrosis. The manufacturer's protocol was followed with some modifications. Briefly, the cells were suspended in 10 μl cold 1× binding buffer in the range of 1×105 to 1×106 cells/ml, and mixed with 10 μl Annexin-V. After incubation at room temperature for 10 min in the dark, the mixtures were added with 380 μl of cold 1× binding buffer and 10 μl of 7-AAD. The stained cells were then analyzed by an EPICS XL apparatus (Beckman Counter) using a single laser emitting excitation light at a wavelength of 488 nm.
10
Annexin V Apoptosis Assay by Flow Cytometry
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The Southern Biotech (Birmingham, AL 35209, USA) ApoScreen™ Annexin V Apoptosis Kit was used. Prior to staining, cells were concentrated to 1 × 106 to 1 × 107 cells/ml. A Vi-Cell XR (Beckman Coulter) was used to assess the cell concentration. Cells were washed in cold PBS twice, after which PBS was removed from the cell pellet. Next, the cells were washed in 1 mL of 1× Annexin V binding buffer once. The supernatants were removed by centrifugation and the cells were suspended in 1× Annexin V binding buffer. 10 μL of FITC-conjugated Annexin V and 10 μL of propidium iodide (PI) were added to the cells. They were then mixed gently and incubated for 15 min on ice in the dark, then diluted with 380 μL of 1× Annexin V binding buffer and analyzed by flow cytometry within 1 h [21 (link),22 (link)].
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