The cultured cells were treated with respective plasmids or oligonucleotides and then treated with cisplatin at a final concentration of 50 μM for 8 hours. After harvesting and the cells were washed twice with PBS. The cells were resuspended in fluorescein isothiocyanate (FITC) -conjugated Annexin-V binding buffer, and 5 ul FITC-conjugated Annexin-V (Invitrogen, Carlsbad, CA, USA) was added to the cell suspension, avoiding light exposure, and maintained at room temperature for 15 mins before adding 5 ul of propidium iodide (PI), and after 10 mins, the cells were analyzed by flow cytometry.
Fitc conjugated annexin 5
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
FITC-conjugated Annexin V is a fluorescently-labeled protein that binds to phosphatidylserine, a cellular membrane component. It is commonly used in flow cytometry and other cell-based assays to detect and quantify apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (22 mentions)
Annexin 5 binding buffer, by Thermo Fisher Scientific (9 mentions)
Facscalibur, by BD (7 mentions)
Propidium iodide, by Merck Group (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Foxp3 staining buffer kit, by Thermo Fisher Scientific (2 mentions)
Zombie yellow fixable viability kit, by BioLegend (2 mentions)
Annexin 5, by Thermo Fisher Scientific (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (2 mentions)
Binding buffer, by BD (2 mentions)
Cell death detection elisa kit, by Roche (2 mentions)
Monodansylcadaverine mdc, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Cell counting kit 8 cck 8, by Apexbio (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
68 protocols using fitc conjugated annexin 5
1
Annexin-V and PI Apoptosis Assay
2
Apoptosis and Cell Viability Assays
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Annexin V–FITC (FITC-conjugated Annexin V, eBioscience) was used to label apoptotic cells. Dead cells were labeled with propidium iodide (PI) (eBioscience). The staining experiment was performed according to the product instructions. Briefly, 1 million cells were washed in cold PBS and suspended in 0.5 mL staining binding buffer. Annexin V–FITC (5 μL) and PI (1 μL), respectively, were added to the cell suspension. Cells were incubated for 15 minutes at room temperature and subjected to flow cytometric analysis. The results were analyzed using FlowJo software. For Cell Counting Kit-8 (CCK-8, APExBIO Technology) assays, MM cells were seeded at 1 × 105 cells/well in triplicate in 96-well plates and incubated at 37°C in 5 % CO2. After incubation for 48 hours, the CKK8 reagent was added to each well and incubated for 2 hours prior to reading absorbance at 450 nm. The following formula was used to calculate cell viability: percentage = OD value of the treatment group/OD value of the control group × 100.
3
Cell Death Induction Analysis by Flow Cytometry
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Cell death induction was quantitatively analyzed via flow cytometry using double staining with FITC-conjugated annexin V (eBioscience, San Diego, CA, USA) and propidium iodide (PI, 1.0 mg/mL, Sigma-Aldrich). SW480 cells were seeded into 24-well plates (7 × 104 cells/well) in 600 µL MEM per well and allowed to settle for 24 h. Afterward, cells were exposed to two or three different concentrations (0.1–100 µM) of the compounds (based on their cytotoxic activity) for 24 h. After treatment, the medium was collected, and cells were washed once with 37 °C warm PBS and harvested. Following trypsinization, the cell suspension was added to the pre-collected medium and cells were centrifuged (300 g, 3 min). The supernatant was removed, and the cell pellet was resuspended with 1.5 µL FITC-conjugated annexin V in 150 µL binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl and 2.5 mM CaCl2) and incubated at 37 °C for 15 min. An amount of 1 µL of propidium iodide (PI, Thermo Fisher) was added shortly before flow-cytometric analysis using a Guava easyCyte 8HT instrument (Merck Millipore, Burlington, MA, USA) with InCyte software. Results were quantified by using the FlowJo software (TreeStar). At least three independent experiments were conducted.
4
Apoptosis and Cell Surface Receptor Assay
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Cells were seeded in 6-well plates at density of 5 x 105 cells per well one day prior to treatment with indicated drugs. At specific time point, cells were harvested by trypsinization. The collected cells were washed once with 1X Annexin V binding buffer (eBioscience, San Diego, CA, USA) and then incubated in the buffer containing FITC-conjugated Annexin V (eBioscience). After incubated for 30 min at room temperature in dark, the cells were washed once with binding buffer and resuspended in 500 µl binding buffer containing propidium iodide solution (PI, 0.5 μg/ml). Annexin V binding and PI infiltration were evaluated by flow cytometry using a FACSCalibur™ (BD Bioscience, Sparks, MD, USA) and analyzed with CellQuest Pro™ software (BD Bioscience). To measure receptor expression on cell surface, cells were harvested by trypsinization at time points as specified in figure legends. The collected cells were incubated in 100 µl phycoerythrin (PE)-conjugated anti-DR4 or anti-DR5 antibodies (eBioscience) at RT for 30 min in dark. A PE-conjugated mouse IgG isotype control (eBioscience) was used as negative control. Fluorescence signals were then acquired on a FACSCalibur™ and analyzed as described above.
5
Apoptosis and Necrosis Quantification in Neutrophils
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Neutrophils, keratinocytes or cell suspensions obtained from murine abscesses (106 cells per reaction tube) were labeled with FITC-conjugated Annexin V (eBioscience) for 15 min at room temperature, subsequently incubated with PI for 15 min at 4°C (BioLegend) and fluorescence was immediately evaluated by flow cytometry using a BD FACSCanto II cytometer. Collected data were analyzed using the FlowJo software. Neutrophils were gated based on their FSC-A/FSC-H profile (28 (link)) (Supplementary Figure 1B Supplementary Figure 1B
6
Multiparametric Flow Cytometry Analysis
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Cells were fixed and permeabilized using the FOXP3 Staining Buffer Kit (eBioscience, San Diego, CA, USA) and were subjected to intranuclear FOXP3 staining according to the manufacturer’s instructions. Dead cells were stained using the Zombie Yellow™ Fixable Viability Kit (BioLegend, San Diego, CA, USA)or FITC-conjugated Annexin V (eBioscience) and propidium iodide (eBioscience) according to the manufacturer’s instructions. 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose), Invitrogen™) were used to check glucose-uptake ability according to the manufacturer’s instructions. The cells were analyzed by flow cytometry using a BD LSRFortessa™ (BD Bioscience, San Jose, CA, USA), and the data were analyzed using FlowJo (Tree-Star version; Ashland, OR, USA).
7
Annexin V Apoptosis Assay for DLBCL
8
Apoptosis Assessment of Spermatozoa
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Fresh spermatozoa and spermatozoa incubated for 24 hr at 25°C were stained for the apoptotic marker phosphatidylserine by use of FITC-conjugated Annexin V (eBioscience) followed by flow cytometric analysis on an LSRII (Becton Dickinson), following manufacturer’s protocol.
9
Annexin V-FITC and PI Staining for Apoptosis
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As mentioned above, 20×104 K562 cells/well from the control and experimental groups were trypsinized, blocked by FBS, washed twice with PBS, collected, resuspended in the binding buffer (Ref No: 00-0055-56, ebioscience) and kept for 20 min in the dark at 4 oC. In the following, cells were incubated with 100 μl binding buffer containing 5 μl of FITC-conjugated Annexin V (Ref No: 11-8005-74, ebioscience) for 15 min at 25 oC. Afterward, cells were washed with binding buffer and exposed to PI solution in 100 μl binding buffer. Flow cytometry was performed by FACSCalibur (BD Bioscience), and the data were analyzed with FlowJo software ver. X.0.7 [24 (link)].
10
Simvastatin Modulates ABCB1 Transport
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SH-SY5Y cells (5 × 105) were treated with simvastatin as indicated in figures, and surface ABCB1 transporter was mapped with MRK16 antibody (1:100 dilution in PBS, 30 min at room temperature; Kamiya Biomedical Company, Seattle, WA, USA) and visualized with the corresponding Alexa Fluor®488-conjugated goat anti-mouse antibody (1:100 in PBS, 30 min at 4 °C; Invitrogen, CA, USA). Alternatively, C219 (Abcam, Cambridge, UK) or p170 (Neomarkers, Fremont, CA, USA) antibodies (1:50 in 10 % FCS with 1 % NaN3 in PBS, 2 h at room temperature) were used for ABCB1 staining in fixed cells and gave similar results. In all FACS experiments, unstained cells and/or cells mapped with mouse IgG2a (Becton Dickinson, Heidelberg, Germany) were used as negative controls to correct for background. The data were processed off-line with Flowing software (www.flowingsoftware.com ).
Apoptosis was determined with biparametric FACS analysis using FITC-conjugated annexin V (Ebioscience, San Diego, CA, USA) and propidium iodide (PI) as previously described (Minichsdorfer and Hohenegger 2009 (link)).
Apoptosis was determined with biparametric FACS analysis using FITC-conjugated annexin V (Ebioscience, San Diego, CA, USA) and propidium iodide (PI) as previously described (Minichsdorfer and Hohenegger 2009 (link)).
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