Cells were harvested, centrifuged and stained with propidium iodide (PI) and Alexa Flour 488 annexin V (Molecular Probes, Eugene), and then were analysed by flow cytometry.
Alexa flour 488 annexin 5
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 annexin V is a fluorescent probe used for the detection of apoptosis. It binds to phosphatidylserine, which is externalized on the surface of cells undergoing apoptosis. The Alexa Fluor 488 dye provides a green fluorescent signal that can be detected using flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Annexin binding buffer, by Thermo Fisher Scientific (3 mentions)
Fxcycle pi rnase, by Thermo Fisher Scientific (2 mentions)
Accuri c6, by Beckman Coulter (1 mentions)
Accuri c6, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Beckman cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
6 protocols using alexa flour 488 annexin 5
1
Apoptosis Detection by Flow Cytometry
2
Apoptosis Assessment by Flow Cytometry
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Cells were harvested, centrifuged and stained with propidium iodide (PI) or PI and Alexa Flour 488 annexin V (Molecular Probes, Eugene), and then were analysed by flow cytometry.
3
Apoptosis Assay with PI and Annexin V
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Cells were either not treated or challenged with PAC or curcumin, and then harvested, centrifuged and stained with propidium iodide (PI) and Alexa Flour 488 annexin V (Molecular Probes, Eugene, OR, USA) as previously described [14 (link)].
4
Apoptosis and Cell Cycle Analysis
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To analyze apoptosis, cells were treated as indicated and allowed to recover for 4 days. Floating cells were collected, and then attached cells were collected with trypsin and combined. After centrifugation and washing, the cells were incubated with Alexa Flour 488 annexin V and 1 µg ml–1 PI in 1× annexin-binding buffer for 15 min in the dark (Thermo). After resuspending in additional binding buffer, the cells were analyzed on an Accuri C6 (Beckman) using FL1 and FL3.
For cell cycle analysis, 23 h after treatment, cells were pulsed with 20 µM EdU and incubated for an additional hour (Thermo). Cells were collected with trypsin, washed with 1% BSA in PBS and then fixed with Click-IT fixative D. After washing with 1% BSA in PBS, the cells were permeabilized with 1× component E for 15 min, before performing Click chemistry with Alexa Flour 488 azide for 30 min in the dark. Cells were washed with 1× component E, and then resuspended in 500 µl FxCycle PI/RNase (Thermo) for 15 min before analyzing on Accuri C6. Standard gating for cells versus debris and singlet was conducted.
For cell cycle analysis, 23 h after treatment, cells were pulsed with 20 µM EdU and incubated for an additional hour (Thermo). Cells were collected with trypsin, washed with 1% BSA in PBS and then fixed with Click-IT fixative D. After washing with 1% BSA in PBS, the cells were permeabilized with 1× component E for 15 min, before performing Click chemistry with Alexa Flour 488 azide for 30 min in the dark. Cells were washed with 1× component E, and then resuspended in 500 µl FxCycle PI/RNase (Thermo) for 15 min before analyzing on Accuri C6. Standard gating for cells versus debris and singlet was conducted.
5
Apoptosis Induction Assay Using Flow Cytometry
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Cells were seeded into 10 cm dishes at a density of 1 × 104 cells/dish; 24 h later, dishes were treated with 1 μM PF-477736 or DMSO vehicle control. After 48 h, cells were trypsinised, harvested (including those in media and PBS washes), counted and 1 × 106 cells were resuspended into 1x annexin-binding buffer (Thermo Fisher Scientific). Then, 2 μL Alexa Flour 488 Annexin V (Thermo Fisher Scientific) and 1 μL 100 μg/mL PI were added to each 100 μL of cell suspension and incubated for 15 min. Single-stained and unstained controls were stained accordingly. Samples were run through the BD LSR Fortessa flow cytometer (Becton Dickinson, USA), recording 10,000 events for each sample. Data were analysed using FlowJo version 10 (FlowJo LLC).
6
Apoptosis and Cell Cycle Analysis
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To analyze apoptosis, cells were treated as indicated, and allowed to recover for 4 days. Floating cells were collected, and then attached cells were collected with trypsin and combined. After spinning down and washing, cells were incubated with Alexa Flour 488 annexin V and 1 μg/ml propidium iodine in 1x annexin-binding buffer for 15 min in the dark (Thermo). After resuspending in additional binding buffer, cells were analyzed on a CytoFLEX S Flow Cytometer (Beckman) using FL1 and FL3. For cell cycle analysis, 23 h after treatment, cells were pulsed with 20 μM EdU (Thermo), and incubated for an additional hour. Cells were collected with trypsin, washed with 1% BSA in PBS, and then fixed with Click-IT fixative D. After washing with 1% BSA in PBS, the cells were permeabilized with 1x component E for 15 min, before performing Click chemistry with Alexa Flour 488 azide for 30 minutes in the dark. Cells were washed with 1x component E, and then resuspended in 500 μl FxCycle PI/RNase (Thermo) for 15 min before analyzing on Beckman CytoFLEX S Flow Cytometer. Preliminary standard gating for cells versus debris and singlet, and analysis of the results, were conducted with FlowJo™ v10.8 Software (BD Life Sciences).
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