Cells were collected and washed using PBS three times after compound or DMSO treatment on different days according to the requirements. For cell cycle detection, cells were fixed by 70% EtOH overnight at 4 oC. PI solution (200 μL) containing RNAase (Thermo) was added, the mixture was incubated for 15 min, then samples were filtered (40–50 μm nylon net) before detection. FITC Annexin V-PI Apoptosis Detection Kit I (BD Biosciences, 556547) was used for apoptosis detection. The procedures were as follows: 50 μL binding buffer, 2.5 μL PI, and 2.5 μL Annexin V were added to resuspended samples for 15 min. Then 150 μL binding buffer were added before centrifugation. Finally, the cells were resuspended in 200 μL binding buffer for FCM analysis. For CD11b (BD Biosciences, 555388), CD14 (BD Biosciences, 555397), CD41b (BD Biosciences, 555469), and CD61 (BD Biosciences, 555754) detection, antibodies were added to cells prior to incubation for 30 min, then they were washed by PBS three times before FCM detection. A mitochondrial membrane potential detection kit (BD Biosciences, USA) was selected for mitochondrial membrane potential detection and used according to the manufacturer’s instructions. FCM assays were conducted on FCM (ACEABIO | NovoCyte or BD FACSCanto, USA). Repeat three times for each sample.
Annexin 5 fitc pi apoptosis detection kit 1
Manufactured by BD
Sourced in United States
The Annexin V-FITC/PI Apoptosis Detection Kit is a laboratory reagent used to detect and quantify apoptosis in cell samples. It contains Annexin V-FITC, a fluorescent conjugate of the Annexin V protein, and propidium iodide (PI), a DNA-binding dye. The kit allows for the identification of early and late apoptotic cells through flow cytometry or fluorescence microscopy analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 cytometer, by BD (2 mentions)
Pi rnase staining buffer, by BD (2 mentions)
Mitochondrial membrane potential detection kit, by BD (1 mentions)
Rnaase, by Thermo Fisher Scientific (1 mentions)
Novocyte, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Flow cytometry, by Thermo Fisher Scientific (1 mentions)
Guava easycyte flow cytometer, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Propidium iodide, by BD (1 mentions)
Stattic, by MedChemExpress (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
C6 plus, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
11 protocols using annexin 5 fitc pi apoptosis detection kit 1
1
Multiparametric Cell Analysis Protocol
2
Apoptosis Analysis of Prostate Cancer Cells
3
Apoptosis and Necrosis in Breast Cancer
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Apoptosis and necrosis of breast cancer cell lines were evaluated by flow cytometry on FACSCanto II cytometer (BD, San Diego, CA, USA). The MDA-MB-231 and ZR-75-1 cells (2.0 × 105 per well) were seeded in 2 mL of medium in six-well plates. After 24 h, the medium was removed, replaced with the rGO suspension in medium, at 50 μg/mL or 100 μg/mL concentrations. All cell lines were incubated for 24 h and 48 h. The cells were detached, resuspended in medium and then in binding buffer. Subsequently, the cells were stained with FITC Annexin V and PI (FITC Annexin V apoptosis detection Kit I, (BD PharmingenTM, San Diego, CA, USA) at room temperature, in the dark, for 15 min. Data were analyzed using FACSDiva software (BD PharmingenTM, San Diego, CA, USA).
4
Flow Cytometric Analysis of Apoptosis and Necrosis
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Apoptosis and necrosis of breast cancer cell lines were evaluated by flow cytometry on an FACSCanto II cytometer (BD, San Diego, CA, USA). MDA-MB-231 and ZR-75-1 cells (2.0 × 105 per well) were seeded in 2 mL of medium in six-well plates. After 24 h, the medium was removed and replaced with the rGO suspension in a medium at 100 μg/mL, MG-132 (5 µM) or a mix consisting of rGO (100 μg/mL) and MG-132 (5 µM). All cell lines were incubated for 48 h. The cells were detached, resuspended in a medium and then resuspended in binding buffer. Subsequently, the cells were stained with FITC Annexin V and PI (FITC Annexin V apoptosis detection Kit I, (BD PharmingenTM, San Diego, CA, USA) at room temperature, in the dark, for 15 min. Data were analyzed using FACSDiva software, Ver. 6.1.3 (BD PharmingenTM, San Diego, CA, USA), and 10.000 cells were measured per sample.
5
Apoptosis Detection via Annexin V-FITC/PI Assay
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An Annexin V-FITC/PI Apoptosis Detection kit I (BD Pharmingen, San Diego, CA, USA) was adopted to examine the cell apoptosis according to the manufacturer's instructions. HCT-8 cells were cultured on a six-well plate (3 × 105 cells/well) and allowed to attach for 24 h before treated with NAT-A (0 and 0.5 μM) for 72 h. Then cells were harvested, washed with PBS and resuspended in 1 × binding buffer. Cell suspension (100 μL) was incubated with 5 μL FITC Annexin-V and 5 μL propidium iodide (PI) for 15 min in the dark. Following the incubation, 400 μL 1 × binding buffer was added into each sample before analysis using flow cytometry (Thermo Fisher Scientific, Eugene, Oregon, USA). The lower left section of fluorocytogram (Annexin V-, PI-) represents the normal cells, lower right section of fluorocytogram (Annexin V+, PI-) represents early apoptosis cells, and upper right section of fluorocytogram (Annexin V+, PI+) represents late apoptosis cells.
6
Evaluating R848-Induced Apoptosis in HKLs
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To evaluate the effect of R848 on HKLs apoptosis, freshly isolated HKLs cultured in 6-well plates (106 cells/well) were treated with CQ (final concentration of 5 μg/ml) for 1 h and then treated with R848 (final concentration of 8 μg/ml) for 24 h; the survival of HKLs was evaluated with the Annexin V-FITC/PI Apoptosis Detection Kit I (BD Pharmingen™, USA) and flow cytometry analysis performed on a Guava easyCyte™ flow cytometer (EMD Millipore Corp., USA). Final data was analyzed with guavaSoft 3.1.1. To examine the requirement for Myd88 in R848-induced HKL apoptosis, 50 μM Pepinh-MYD or Pepinh-Control was incubated with HKLs for 6 h at 26°C. After the incubation, the cells were treated with R848 (final concentration of 8 μg/ml), and flow cytometry was used as described above to detect cell survival. To analyze the impact of NF-κB activation on R848-induced apoptosis in HKLs, 1 μM BAY-11-7082 was added to cells for 1 h at 26°C. Then, the cells were treated with R848 (final concentration of 8 μg/ml). The experiments were repeated in triplicate.
7
Apoptosis Induction in MDA-MB-231 Cells
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MDA-MB-231 cells were treated with different concentrations of the complexes (0, 10, 20, and 40 μM) for 72 h, then collected and stained with an Annexin V-FITC/PI Apoptosis Detection Kit I (BD, 556547). The MDA-MB-231 cells were fixed with 70% ethanol at 4 °C for 30 min, and then collected and stained with PI/RNase Staining Buffer (BD, 550825). The cells were analyzed by flow cytometry (Beckman, CytoFLEX). All experiments were repeated three times.
8
Cell Cycle and Apoptosis Analysis
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To verify the effects on cell cycle distribution and the percentage of apoptotic cells by SchA, cells were examined by flow cytometry. For cell cycle analysis, A549 and H1975 cells were incubated with SchA or DMSO for 24 hours, and then fixed in pre‐chilled 70% ethanol overnight. Fixed cells were washed with PBS and stained with propidium iodide (PI)/RNase staining buffer (#550825, BD, San Jose, CA, USA). Subsequently, cell cycle distribution was measured on flow cytometer (Beckman Coulter, Inc., California, USA).
Cell apoptosis samples were prepared using Annexin V‐FITC/PI Apoptosis Detection Kit I (#556547, BD, San Jose, CA, USA) and quantified by flow cytometer (Beckman Coulter, Inc., California, USA). The flow cytometric results were visualized with FlowJo 10 (BD, San Jose, CA, USA).
Cell apoptosis samples were prepared using Annexin V‐FITC/PI Apoptosis Detection Kit I (#556547, BD, San Jose, CA, USA) and quantified by flow cytometer (Beckman Coulter, Inc., California, USA). The flow cytometric results were visualized with FlowJo 10 (BD, San Jose, CA, USA).
9
Annexin V-FITC/PI Apoptosis Assay
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Apoptotic cells were labeled with the Annexin V-FITC/PI Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) and quantified on flow cytometry. Briefly, cells were harvested using trypsin/EDTA and washed with PBS. After centrifuge, cell pellets were re-suspended in the staining buffer with the addition of FITC-labeled Annexin V and PI and kept in the dark for 10–15 min at room temperature. Finally, the apoptotic cells were detected by a FACScan Flow Cytometer (Becton Dickinson, Franklin lakes, NJ, USA). We also studied whether lncRNA HAR1A regulates the proliferation of NSCLC cells through the STAT3 signaling pathway. Cells infected with lenti-sh HAR1A were treated with Stattic, an inhibitor of STAT3 (MedChemExpress LLC, Princeton, NJ, USA), for 24 h.
10
Evaluating Cell Apoptosis via Annexin V-FITC/PI Assay
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C4-2B and LNcaP cell lines were collected from 6-well plate after 72 h siRNA transfection with trypsinization. Then, cells was washed with PBS for 2 times, and resuspended in 1× staining buffer at a concentration of 1–5 × 106 cells per ml. Cell apoptosis was evaluated by AnnexinV-FITC/PI Apoptosis Detection Kit I (BD, USA), according its manufacturer’s protocol. Briefly, the negative control siRNA transfection cells were quadripartition: neither PI nor AnnexinV-FITC, only 5 μl PI stained, only 10ul AnnexinV-FITC stained or both 5 μl PI and 10 μl AnnexinV-FITC. In addition, 5 μl PI and 10 μl AnnexinV-FITC were also stained in the ADSL siRNA transfection cells, which were incubated 15 min in the dark on the ice. Cell apoptosis was then detected by flow cytometry (BD C6 Plus, USA) with Cellquest software (BD Biosciences, Franklin Lakes, NJ, USA).
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