Following transfection of HCT116 cells with calnexin or control scramble siRNA for 24 h and subsequent treatment with 5FU or vehicle for 48 h, cells were harvested and the induction of cell death was assessed by Annexin V and PI staining using flow cytometry. Cells were incubated at room temperature in binding buffer (10 mM HEPES, 135 mM NaCl, 5 mM CaCl2) which contained an Annexin V-FITC conjugate (1 μl/ml; BioVision, Mountain View, CA, USA) and propidium iodide (PI; 1 μl/ml, BioVision) for 15 min. Cells were counted in a Cyflow ML 16 flow cytometer (Partec, Münster, Germany). Excitation of Annexin V-FITC was done with a 488 nm laser and fluorescence emission was collected in the FL1 channel through a 520 nm band pass filter. PI was excited with a 488 nm laser and fluorescence emission was collected in the FL3 channel through a 620 nm long pass filter. 1 × 104 gated cells were acquired for each sample and analyzed using the Flowmax software (Partec).
Annexin 5 fitc conjugate
Manufactured by Abcam
Sourced in United States
Annexin V-FITC conjugate is a fluorescently labeled protein that binds to phosphatidylserine (PS), a phospholipid exposed on the surface of cells undergoing apoptosis. The FITC (fluorescein isothiocyanate) label allows for the detection and quantification of apoptotic cells using techniques such as flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (3 mentions)
Annexin 5 fitc apoptosis kit, by Abcam (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Lsr 2, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Q vd oph, by Selleck Chemicals (1 mentions)
5 protocols using annexin 5 fitc conjugate
1
Calnexin Knockdown Enhances 5FU-Induced Cell Death
2
Enolase-Induced Apoptosis in IEC-6 Cells
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IEC-6 cells were grown on coverslips in DMEM at 37°C and 5% CO2 and when confluence was reached, the monolayers were exposed to purified rGd-eno at a final concentration of 100 μg/mL (reference glycolytic activity 58 μmol/min xmg of protein) for 2 h. Negative controls were IEC-6 cells incubated with DMEM alone and positive controls were IEC-6 cell monolayers exposed to 50 µM H2O2, cells without enolase were then incubated for 2h under the same conditions. After this, coverslips were treated with Annexin V-FITC conjugate (BioVision, Milpitas, Ca, USA) for 5 min following the manufacturer´s instructions, fixed with 2% PFA for 1 h, washed once with PBS and analyzed using an Axioscope Zeiss Zeiss epifluorescence microscope. For quantification by flow cytometry, cells were collected and exposed to the treatments mentioned above an analyzed with the Annexin V-FITC Apoptosis Kit (BioVision, Milpitas, Ca, USA) following the manufacturer’s instructions.
3
Quantifying Glioblastoma Cell Death
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Cell death was measured using a BD LSRII flow cytometer. Recurrent GBM cells were seeded as gliomaspheres, 300,000 cells per well in 6-well plates. After 24 h cells were either pretreated with 150 μM TMZ for 24 h followed by treatment with 3 μM CYC065 or 100 nM THZ1 for 120 h or treated with 3 μM CYC065 and 100 nM THZ1 as single agents for 120 h. Following treatments, gliomaspheres were pelleted, dissociated using Accutase and were next incubated in 100 μL of binding buffer (10 nM HEPES, 135 nM NaCl, 5 mM CaCl2) containing AnnexinV-FITC conjugate (1:200, BioVision, Mountain View, CA, USA) and propidium iodide (1:500, Sigma-Aldrich, Arklow, Ireland) for 10 min on ice in the dark. FITC was excited at 488 nm and fluorescence emission was collected in the FL1 channel through a 520 nm band-pass filter. PI was excited at 561 nm and fluorescence emission was collected through a 605/40 nm band-pass filter and a 570 nm long pass filter. A total of 1 × 104 gated cells were acquired. Acquired data from the flow cytometry analyses were analysed using FlowJo Software.
4
Annexin-V assay for apoptosis analysis
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Cells were seeded at a density of 50,000 cells/well in a 24-well plate and incubated for 24 h before treatment. Where Q-VD-OPh was used to assess the requirement for caspase activity in cell death, cells were pre-treated with 30 µM Q-VD-OPh for 30 min before additional agents were applied. Unless otherwise stated, Daoy, ONS76, UW228, D425-Med and D458-Med cells were treated for 48 h, and D283-Med cells for 72 h, due to their increased doubling time. Following treatment, adherent cells were detached using Trypsin-EDTA (Sigma-Aldrich), pelleted, and washed, while suspension cells were pelleted, washed and clusters were manually dissociated using a pipette. Cells were incubated in 100 µL Annexin-V binding buffer (10 nM HEPES, 135 nM NaCl, 5 mM CaCl2, pH 7.4) containing Annexin-V-FITC conjugate (1:200) (BioVision, Mountain View, CA, USA) and propidium iodide (1:500) (Sigma-Aldrich) for 20 min in the dark. Cells were analysed using a BD LSRII flow cytometer (BD Biosciences, Oxford, UK), and acquired data was analysed using Flowing Software Version 2.5.1 (Turku Bioscience, Tykisokatu 6, FI-20520 Turku, Finland).
5
Measuring Cell Death in Glioblastoma
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Cell death was measured using a BD LSRII flow cytometer (BD Biosciences, Oxford, UK). U87 and U343 cells were plated as monolayers in a 6-well plate (500 000 cells/well). GBM PDCLs were seeded as gliomaspheres, 300 000 cells per well in a 6-well plate. After 24 h cells were treated with DMSO (0.1%), 3 μM CYC065 or 100 nM THZ1 for 72 or 120 h. For caspase dependence experiments, cells were pre-treated with 50 μM QVD-OPh (Selleckchem, Houston, TX, USA) for 1 h and then treated with 3 μM CYC065 or 100 nM THZ1 for 72 h. U87 and U343 cells were detached following 72 h treatment using Trypsin-EDTA solution (Sigma-Aldrich, Arklow, Ireland) for 4 min at 37 °C and 5% CO2. Following treatments, gliomaspheres were pelleted, dissociated using Accutase (Thermo Fisher Scientific, Waltham, MA, US). Cells were next incubated in 100 μL of binding buffer (10 nM HEPES, 135 nM NaCl, 5 mM CaCl2, pH 7.4) containing AnnexinV-FITC conjugate (1:200) (BioVision, Mountain View, CA, USA) and propidium iodide (1:500) (Sigma-Aldrich, Arklow, Ireland) for 20 min on ice in the dark. A total of 1 × 104 gated cells were acquired. Acquired data from the flow cytometry analyses were analysed using FlowJo Software 10.6.2. Version 5.0 (Becton, Dickinson and Company, Franklin Lakes, NJ, US).
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