Cells were seeded overnight and incubated with colchicine (10 nM), paclitaxel (10 nM), and CH-2-77 (2.5 nM, 5 nM, and 10 nM) for 24 h. Then, cells were digested with trypsin-EDTA (#2520056, Gibco) and stained with Annexin-V-FITC (eBioscience, 5 μL) and propidium iodide (PI, 10 μL) in Annexin-V-FITC binding buffer (100 μL) for 10 min. Apoptotic cells were detected by flow cytometry. For cell cycle distribution, cells with the same treatments as the apoptosis assay were harvested, washed, and fixed. After permeabilization, cells were stained with rabbit anti-phospho-histone H3 (Ser 10) antibody (#9701, 1:50, CST) for 1 h at room temperature followed by a 30 min incubation with Alexa Fluor 488 goat anti-rabbit secondary antibody (# A-11008, 1:50, Molecular Probes). After washing 2 times with PBS, cells were resuspended in PI/RNase staining solution and detected by cytometry after a 5 min incubation. Cells in sub-G1, G1, S, and G2 phases were gated by a reported method (18 (link)). The experiment was conducted using three biological replicates with three technical replicates per treatment per cell line.
Annexin 5 fitc
Manufactured by Thermo Fisher Scientific
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Annexin V-FITC is a fluorescently labeled protein used for the detection and quantification of cells undergoing apoptosis. It binds to phosphatidylserine, a phospholipid that is exposed on the cell surface during the early stages of apoptosis. The FITC fluorescent label allows for the visualization and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (95 mentions)
Facscalibur, by BD (33 mentions)
Propidium iodide, by Merck Group (27 mentions)
Facscanto 2, by BD (16 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Facscalibur flow cytometer, by BD (13 mentions)
7 aad, by BD (13 mentions)
Binding buffer, by Thermo Fisher Scientific (12 mentions)
Annexin 5 binding buffer, by Thermo Fisher Scientific (12 mentions)
Cellquest software, by BD (11 mentions)
Facscan, by BD (10 mentions)
Rpmi 1640, by Thermo Fisher Scientific (9 mentions)
Annexin 5 fitc, by BD (8 mentions)
Facscan flow cytometer, by BD (8 mentions)
Accuri c6, by BD (8 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (7 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Propidium iodide, by BD (7 mentions)
Lipopolysaccharides (lps), by Merck Group (7 mentions)
Lsrfortessa flow cytometer, by BD (7 mentions)
503 protocols using annexin 5 fitc
1
Apoptosis and Cell Cycle Analysis
2
Apoptosis Assay for Melanoma Cells
3
Annexin V-FITC Cell Death Assay
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Tumor cell death induced by AmB was monitored after incubation with indicated drug concentrations for indicated time periods. Dox at ICD inducing concentration (5 μmol) served as a positive control. Cisplatin (non-ICD inducer at apoptosis inducing low and high concentrations) was used as a negative control in some experiments.
Cell death was assessed by annexin V FITC (Life Technologies Cat# V132245) staining. Briefly, 10^6 cells per sample were collected, washed in PBS (Gibco Cat# 14190–094), pelleted and resuspended in an incubation buffer containing annexin V-FITC antibodies. The samples were kept in the dark and incubated for 15 min prior to the addition of DAPI (Sigma Cat# D9542), and subsequent analysis was performed with a FACSCanto II (BD Bioscience).
Cell death was assessed by annexin V FITC (Life Technologies Cat# V132245) staining. Briefly, 10^6 cells per sample were collected, washed in PBS (Gibco Cat# 14190–094), pelleted and resuspended in an incubation buffer containing annexin V-FITC antibodies. The samples were kept in the dark and incubated for 15 min prior to the addition of DAPI (Sigma Cat# D9542), and subsequent analysis was performed with a FACSCanto II (BD Bioscience).
4
Evaluating Apoptosis in HAP1 Knockdown Cells
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SEM cells (#; 1 × 104) stably infected with retrovirus carrying either pRS empty vector (#+pRS) or pRS-shHAP1 (#+pRS-shHAP1) were seeded in 96-well plates coated with 0.02% poly-L-ornithine. Cells pre-treated with 2 μg/mL D,L-methadone or 2 μM 14–22 amide (myr), for 8 h were stained with 0.5 μg/mL annexin V-FITC (Invitrogen) and visualized at λex = 485 nm and λem = ×530 nm and ×10 magnification using an IX71 Olympus inverted microscope (Tokyo, Japan). The percentage of apoptotic cells was calculated based on a total of 101–298 cells counted per treatment. Analysis was performed using ImageJ 1.4.1 (NIH, United States). For flow cytometry, cells (0.5 × 106) pre-treated with 2 μM 14–22 amide (myr) then treated with 2 μg/mL D,L-methadone for 12 h were harvested, washed twice with 1× PBS, stained with annexin V-FITC (2 μL) and propidium iodide (2 μL), and analysed using an Attune NxT flow cytometer (ThermoFisher Scientific, United States).
5
Apoptosis Analysis by Flow Cytometry
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The cells in each group were cultured for 48 h, treated with 0.25% trypsin for 24 h, and then washed with phosphate buffer (PBS) for 3 times. Add 195 μL Annexin V-FITC (Invitrogen, MA, USA) binding solution to gently suspend the cells, then add 5 μL Annexin V-FITC and mix well; finally add 10 μL PI staining solution to mix well. After mixing, the cells were placed at room temperature in dark for 15 min (the cells were resuspended for 3 times), and then placed in ice bath; the apoptosis rate was detected by flow cytometry.
6
Measuring Apoptosis in hVSMCs Using FCM
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Flow cytometry (FCM) analysis was performed to measure apoptosis in hVSMCs. Cells were resuspended in 1x buffer, and 195 μL Annexin V-FITC binding buffer was added after the supernatant was removed. Then, 5 μL Annexin V-FITC (Thermo Fisher Scientific, MA, USA) was added to the suspension and incubated at room temperature for 10 min in the dark. The supernatant was removed after incubation, and 190 μL Annexin V-FITC binding buffer was added to resuspend the cells, and then 10 μL propidium iodide staining solution (Thermo Fisher Scientific) was added. The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, NJ, USA), and the data were analyzed using FlowJo software.
7
Apoptosis Assay using Flow Cytometry
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Cells were seeded in 24-well plate at a concentration of 0.2 million cells per well, and allowed to attach by 24-hour incubation. The treatment with the test compounds was carried out for 48 hours and the cells were harvested by trypsinization, followed by washing with PBS twice to remove the media traces by centrifugation at 2,500 rpm at 4 ºC for 10 min each. The cell pellet was resuspended in 500 μL of the annexin V-FITC/ PI buffer (10X buffer = 0.1 M HEPES, 25 mM CaCl2 NaCl 1.4 M dissolved in 1X PBS), followed by the addition of annexin V-FITC (Cat # A13199, Thermo Fisher Scientific, Waltham, MA USA 02451) and PI (0.2 ng/500 μL of binding buffer) 5 μL, and 1 μL, respectively, into each sample. Samples were incubated for 15 min, and proceeded for apoptosis analysis on flow cytometer. PI with excitation/emission maxima of 493/636 nm, was detected on FL2 channel, while annexin V-FITC with excitation/emission maxima of 494/518 nm, was detected on FL1 channel. Live cells, PI positive cells (dead cells), and annexin V-FITC positive cells (cells were treated with camptothecin) were used as negative controls to calibrate the FL2, and FL1 channels, respectively.
8
Annexin V and PI Staining for Sperm Viability
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FITC-Annexin V (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect PS translocation and combined with PI to differentiate between membrane-intact and damaged cells, with or without PS translocation (Pena ˜ et al. 2003) . For this analysis, aliquots of 350 μL (cells diluted 1:75 in Annexin-binding buffer, Invitrogen, Waltham, MA, USA) were stained with 2 μL FITC-Annexin V and 3 μL 1.5 mM PI. After incubation at 37°C in darkness for 10 min, samples were evaluated by flow cytometry. The monitored parameters were FS log, SS log, FL1 log (FITC-Annexin V) and FL4 log (PI). The percentage of viable spermatozoa without PS translocation was considered.
9
Quantifying Apoptosis by Flow Cytometry
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Per well, 105 cells were seeded in 6-well plates in 2 ml medium each. After 24 h, cells were trypsinised and washed with PBS. Cells were resuspended in 100 µl of AnnexinV Binding Buffer (BD Pharmingen) containing 5 µl of AnnexinV-FITC (eBioscience) and incubated for 15 min on ice in the dark. After washing with PBS, 100 µl of AnnexinV Binding Buffer containing 2.5 µl of 7-AAD (BD Pharmingen) was added and the samples were incubated for 10 min at room temperature. FACSCanto II with FACSDiva software was used for flow cytometry and FlowJo VX was used for data analysis.
10
Annexin V-FITC Apoptosis Assay
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Apoptotic events were measured by double staining with Annexin V-FITC and propidium iodide according to the manufacturer’s protocol (Annexin V-FITC apoptosis detection kit, eBioscience, San Diego, CA, USA). The cells were incubated at 37°C with culture medium containing cetuximab or the synthesized SPIONs for 24 hours as indicated. The cell populations were analyzed on a FACScan flow cytometer using the CELLQuest® flow analysis software.
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