Sixteen hours after transfection, NB cells were treated with perifosine (AS, 7.5 μM; BE2, 10 μM), MK-2206 (AS, 10 μM; BE2, 7.5 μM), etoposide (AS, 1 μg/ml; BE2, 4 μg/ml), or cisplatin (AS, 1 μg/ml; BE2, 2 μg/ml), respectively, for 48 h. Apoptotic cells were detected by flow cytometry using Annexin V staining and PI staining. In detail, all cells in plate were collected, then washed with PBS once. Then, they were suspended in 1× Annexin V binding solution (Dojindo, Japan), diluted to 1 × 105 cells/ml, and transferred to a new tube. Then 5 μl of Annexin V or PI staining solution or both were added to the EP tubes and incubated in dark conditions for 15 min at room temperature. Finally, Annexin V/PI flow cytometry was performed as the manufacturer’s instruction.
Annexin 5 binding solution
Manufactured by Dojindo Laboratories
Sourced in Japan
1× Annexin V binding solution is a reagent used in cell biology research. It provides a buffer for the binding of Annexin V, a protein that specifically binds to phosphatidylserine, a marker of apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest pro software, by BD (2 mentions)
Microplate spectrophotometer, by Bio-Rad (2 mentions)
Annexin 5 fitc, by Dojindo Laboratories (2 mentions)
Propidium iodide solution, by Dojindo Laboratories (2 mentions)
Cck 8 kit, by Dojindo Laboratories (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Rnase a, by Dojindo Laboratories (1 mentions)
6 protocols using annexin 5 binding solution
1
Apoptosis Analysis in NB Cells
2
Quantifying Apoptosis in TNC Knockdown Cells
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Stably transfected TNC knockdown cells (PC-1.0) and shRNA-TNC transfected cells (AsPC-1) were harvested and incubated with 1×Annexin V binding solution (Dojindo, Kumamoto, Japan). Then the cells were stained with propidium iodide (PI) and Annexin V. Cellular apoptosis was detected and analyzed with a FACS Calibur flow cytometer using CellQuestPro software (Becton Dickinson, Franklin Lakes, NJ, USA).
3
Annexin V-PI Flow Cytometry
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After transfection for 48–72 h, cells were harvested and incubated with 1× annexin V binding solution (Dojindo). According to the manufacturer’s instructions, cells were stained with PI or annexin V and then analyzed with a FACScalibur flow cytometer using CellQuestPro software (Becton Dickinson).
4
Cell Proliferation and Apoptosis Assays
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Cell proliferation was evaluated at indicated time points using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer’s protocol. CCK-8 reagent (10%) was added to each well for 3 h at 37 °C. Viability was evaluated by measuring the absorbance at a 450-nm wavelength with using a microplate spectrophotometer (BioRad).
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 1000 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. The apoptosis assays were run and analyzed with BD Jazz.
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 1000 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. The apoptosis assays were run and analyzed with BD Jazz.
5
Annexin V and PI Staining for Cell Apoptosis
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BMDMs were seeded on the samples for 3 days. The cells were detached by Trypsin-EDTA and resuspended using 10-fold diluted Annexin V Binding Solution (Dojindo, AD10, Japan) to make a final cell concentration of 1 × 106 cells/ml. Then, the cells were added with Annexin V. After incubating for 10 min, the cell suspension was added with 5 μl of propidium iodide (PI) solution. This solution was detected using FACScan flow cytometer detection (Beckman, USA). The result of cytometric detection was calculated by Flow Jo software.
6
Cell Proliferation and Apoptosis Assays
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Cell proliferation was determined at indicated time points using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer's protocol. We added 10% of CCK-8 solution to each well for 3 h before measuring the absorbance at 450 nm using a microplate spectrophotometer (Bio-Rad).
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 100 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. For cell cycle assays, cells were fixed in 70% precooled ethanol on ice for 2 h, centrifuged at 100 rpm for 5 min, and resuspended with 400 μl PI and 100 μl RNaseA (Dojindo). After a 30 min incubation at 4°C, cells were washed and resuspended with PBS. Both the apoptosis assays and cell cycle assays were run and analyzed with BD Jazz.
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 100 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. For cell cycle assays, cells were fixed in 70% precooled ethanol on ice for 2 h, centrifuged at 100 rpm for 5 min, and resuspended with 400 μl PI and 100 μl RNaseA (Dojindo). After a 30 min incubation at 4°C, cells were washed and resuspended with PBS. Both the apoptosis assays and cell cycle assays were run and analyzed with BD Jazz.
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