For the apoptosis assay, cells were seeded at 18 × 103 cells/250 µl medium/0.95 cm2 growth area in 48-well plates, grown overnight, and treated with morin or cisplatin for 24 h and/or 48 h. Concentrations of drugs’ solvents were corrected in all wells (including control wells) to the constant level, corresponding to the highest used concentration of a particular solvent. After the treatments, the assay was performed using FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI (Invitrogen™) according to the manufacturer’s protocol. The cells were counted with a fluorescence microscope Eclipse Ti (Nikon Instruments Inc.) For each well, the ratio of apoptotic cells to the total number of cells in three different fields was calculated. Samples were prepared in triplicate.
Fitc annexin 5 dead cell apoptosis kit with fitc annexin 5 and pi
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI is a laboratory product designed for the detection and quantification of apoptosis and cell death. The kit includes FITC annexin V and propidium iodide (PI) reagents for flow cytometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti, by Nikon (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Accuri c6, by BD (2 mentions)
Facscan, by Beckman Coulter (1 mentions)
Annexin 5 fitc antibody, by Thermo Fisher Scientific (1 mentions)
Facsarray flow cytometer, by BD (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Z vad fmk caspase inhibitor, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
7 protocols using fitc annexin 5 dead cell apoptosis kit with fitc annexin 5 and pi
1
Apoptosis Assay for Cell Viability
2
Measuring Apoptosis in HaCaT Cells
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After desired treatments, percentage of apoptotic cells was analyzed using double staining with FITC annexin V and propidium iodide (PI). In brief, after different treatment, transfected or untransfected HaCaT cells were collected after trypsin digestion. Then, after rinsing by phosphate-buffered saline (PBS), cells were resuspended in annexin-binding buffer from a FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI for flow cytometry (Invitrogen, Carlsbad, CA, USA). According to the manufacturer’s instructions, cells (100 μL) were stained with 5 μL FITC annexin V and 0.1 μg PI at room temperature for 15 min in the dark. Afterward, stained cells were detected by a FACS can (Beckman Coulter, Fullerton, CA, USA). The percentage of apoptotic cells was analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).
3
Annexin V-FITC Apoptosis Assay
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72 h after transfection with siRNA (siScr or si3e) and 48 h after ionizing radiation (IR) treatment (5 Gy) or CoCl2 treatment (100 μM), cells were collected, washed in Phosphate-Buffered Saline (PBS) and incubated in 100 μl of Annexin-binding buffer 5X (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), containing 5 μl of Annexin V-FITC antibody and 1 μl of Propidium Iodide (PI; Invitrogen Life Technologies, Carlsbad, CA, USA) solution at 100 μg/ml (FITC Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and PI; Invitrogen Life Technologies, Carlsbad, CA, USA) during 15 min at room temperature in the dark. 400 µl of Annexin-binding buffer 5X were then added after washes with PBS–bovine serum albumin 1%. Labeled cells were preserved on ice and run on a flow cytometer (BD Accuri™ C6, BD Biosciences, Franklin Lakes, NJ, USA).
4
Apoptosis and Necrosis Evaluation in Prostate Cancer Cells
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For flow cytometry analysis, 1x105 DU145 and PC3 cells were cultured in 12-well plates at a density of 5x105 cells per well. The culture medium was replaced after 24 h, and transfection with miR-185-5p, miR-1266-5p and miR-34 was performed. The cells transfected with blank Lipofectamine® 2000 were considered as negative controls.
To evaluate the extent of apoptosis and necrosis, the FITC Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and PI (Invitrogen) (for DU145) or the PE Annexin V Apoptosis Detection Kit I (BD Pharmingen, Stockholm, Sweden) (for PC3) were used 24 h after cell transfection. A cell pellet was obtained from the centrifuged harvested cells of each well and washed with cold PBS, followed by addition of 5 µl Annexin V and 1 µl of the 100 µg/ml PI (for DU145), or 5 µl PE Annexin and 5 µl 7-ADD (for PC3), to 100 µl of cell suspension containing 1x105 cells. Following incubation for 15 min at 25 °C, 400 µl binding buffer was added to each tube for analysis using a FACS Array flow cytometer (Becton Dickinson, San Jose, CA, USA) at the indicated wavelengths (FITC: excitation/emission wavelength, 550/573 nm; 7-ADD: excitation/emission wavelength, 488/674 nm).
To evaluate the extent of apoptosis and necrosis, the FITC Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and PI (Invitrogen) (for DU145) or the PE Annexin V Apoptosis Detection Kit I (BD Pharmingen, Stockholm, Sweden) (for PC3) were used 24 h after cell transfection. A cell pellet was obtained from the centrifuged harvested cells of each well and washed with cold PBS, followed by addition of 5 µl Annexin V and 1 µl of the 100 µg/ml PI (for DU145), or 5 µl PE Annexin and 5 µl 7-ADD (for PC3), to 100 µl of cell suspension containing 1x105 cells. Following incubation for 15 min at 25 °C, 400 µl binding buffer was added to each tube for analysis using a FACS Array flow cytometer (Becton Dickinson, San Jose, CA, USA) at the indicated wavelengths (FITC: excitation/emission wavelength, 550/573 nm; 7-ADD: excitation/emission wavelength, 488/674 nm).
5
Apoptosis Measurement by Flow Cytometry
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Apoptosis of cells (100,000 cells/well) was measured using FITC Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and PI (Invitrogen, Carlsbad, CA, USA) 72 h after transfection. Harvested cells were centrifuged and suspended in 1× Annexin binding buffer. Cell suspension was incubated with Annexin V-FITC and PI according to manufacturer’s protocol. Samples were analyzed using Accuri C6 flow cytometer (BD Biosciences, Erembodegem, Belgium). Cells were discriminated into viable (both annexin V-FITC/ PI negative), apoptotic (annexin V-FITC positive), and dead cells (both annexin V-FITC/ PI positive).
6
Annexin V/PI Apoptosis Assay with siRNA
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Seventy-two hours after transfection with siRNA (siScr or siInt6) and treatment or not with 30 μM of Z-VAD-FMK caspase inhibitor (Sigma Aldrich, Saint-Louis, MO, USA), cells were collected, washed in PBS and incubated in 100 μL of Annexin-binding buffer 5× (10 mM HEPES pH7.4, 140 mM NaCl, 2,5 mM CaCl2), containing 5 μL of Annexin V-FITC antibody and 1 μL of Propidium Iodide (PI, Invitrogen Life Technologies, Carlsbad, CA, USA) solution at 100 μg/mL (FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, Invitrogen Life Technologies, Carlsbad, CA, USA) during 15 min at room temperature (RT) in the dark. Four hundred microliters of Annexin-binding buffer 5× were then added after washes with PBS-BSA 1%. For cell cycle analysis, cells were fixed with cold ethanol 100% and then permeabilized with Triton ×100 at 0.25%. Cells were then labeled with Ki67 (Abcam, Paris, France) during 45 min at RT and treated with RNAse 1 μg/mL before labeling with PI during 2 hrs at RT. Labeled cells were preserved on ice and run on a flow cytometer (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, USA).
7
Apoptosis Assay for Drug Evaluation
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For the apoptosis assay, cells were seeded at 18 × 103 cells/250 μL medium/0.95 cm2 growth area in 48-well plates, grown overnight, and treated with Que or Doc for 24 h and/or 48 h. The concentrations of the drug solvents were corrected in all wells (including the control wells) to a constant level corresponding to the highest used concentration of a particular solvent. After the treatments, the assay was performed using the FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI (Invitrogen™, Grand Island, NY, USA) according to the manufacturer’s protocol. The cells were counted with a fluorescence microscope Eclipse Ti (Nikon Instruments Inc., Melville, NY, USA). For each well, the ratio of apoptotic cells to the total number of cells in three different fields was calculated. The samples were prepared in triplicate.
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