For flow cytometry, PE or FITC-labeled anti-human CD4, CD8a, PE-labeled anti-human CDw210 (IL-10 receptor α), FITC-labeled Annexin V (BD Biosciences, San Jose, CA), Alexa Fluor 488-labeled anti-human Ki-67 (Biolegend, San Diego, CA), PE-labeled anti-human IRF4 (eBioscience, San Diego, CA), and their isotype controls were used. To detect HTLV-1 Tax, Alexa Fluor 488-conjugated Lt-4 [76 (link)] and its isotype control (mouse IgG3) antibodies were used. For immunoblotting assays, antibodies specific for phospho-NF-κB p65 (Ser536), NF-κB p65, phospho-NF-κB p100 (Ser866/870), NF-κB p100/p52, cleaved caspase-3, caspase-3, survivin, phospho-STAT3 (Tyr705), STAT3 and IRF4 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for α-tubulin and β-actin were obtained from Sigma-Aldrich (Buchs, Switzerland). AS101, the non-toxic tellurium IL-10-inhibitor (Tocris, Ellisville, MO) [47 (link)] and cucurbitacin I (JSI-124), a STAT3-inhibitor (Tocris) [48 (link)] were also used.
Fitc labeled annexin 5
Manufactured by BD
Sourced in United States, Japan
FITC-labeled Annexin V is a fluorescently labeled version of the Annexin V protein, which binds to phosphatidylserine residues exposed on the surface of apoptotic cells. This can be used to detect and quantify apoptosis in cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by BD (7 mentions)
Accuri c6, by BD (4 mentions)
Rnase a, by Merck Group (3 mentions)
Uorescence microscope, by Nikon (3 mentions)
Cell light edu dna cell proliferation kit, by Keygen Biotech (3 mentions)
Facscalibur, by BD (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Pi apoptosis detection kit, by BD (2 mentions)
Facscalibur system, by BD (2 mentions)
Facs c6, by BD (2 mentions)
100 μl binding buffer, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
As101, by Bio-Techne (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pyronin y, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
47 protocols using fitc labeled annexin 5
1
Multiplex Flow Cytometry and Immunoblotting Assay
2
Multiparametric Flow Cytometry Analysis
3
Annexin V-FITC/PI Apoptosis Assay
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Apoptosis in IEC-6 cells was identified using the FITC-labeled AnnexinV/propidium iodide (PI) Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ) following the manufacturer's instructions. After HR, the IEC-6 cells were suspended in 200 ml of 1 × binding buffer (1 × 10 6 cells/ml). The cells were incubated with AnnexinV (1:20) for 3 min followed by PI for 15 min in the dark at room temperature. The cells were subjected to flow cytometry using the BD FACS Calibur system, and the data were analyzed using FlowJo software.
4
DEH Exposure Impacts Cell Cycle and Apoptosis
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Cells were placed into a cell culture plate and cultured under standard conditions. The adherent cells were incubated with cell-culture medium containing different concentrations of DEH for 2 days for analysis of the cell cycle and apoptosis. For the cell cycle assay, the cells were harvested and fixed in cold 75% ethanol at 4 °C overnight. After washing with PBS to remove the residual alcohol, the cells were incubated with propidium iodide (PI; BD, USA) and RNase A (Sigma Aldrich, USA) at 37 °C for 1 h. For the apoptosis assay, cells were harvested and washed with cold PBS, and then resuspended in 100 μL binding buffer (BD, USA). Thereafter, the cells were incubated with FITC-labeled Annexin V (BD, San Jose, CA, USA) and PI at room temperature for 15 min. All the samples were analyzed by the FACS C6 (BD, USA) using Cell Quest software.
5
Apoptosis Detection by Flow Cytometry
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Flow cytometry was performed using propidium iodide (PI) and fluorescein isothiocyanate (FITC)-labeled annexin V (BD Pharmingen, San Diego, CA) to detect phosphatidylserine externalization as an endpoint indicator of early apoptosis. 24 h after the treatments, the cells were washed twice with cold PBS at 4 °C and centrifuged at 1000×g for 5 min. 5-μl FITC-labeled Annexin V, 10-μl PI, and 5-μl Hanks' balanced salt solution were added to the cell suspension and mixed gently. After 30-min incubation in the dark, the cells were analyzed by flow cytometry (BD Bioscience, USA).
6
Scanning Electron Microscopy and Cell Cycle Analysis
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Cells were fixed with PBS-based 4% paraformaldehyde for 20 min, treated with ethanol, and dehydrated for 5 min. After being treated with hexamethyldisilazane (Sigma–Aldrich, St. Louis, MO, USA), they were coated with gold and observed by a scanning electron microscope, JSM-7600F (Jeol, Akishima, Japan). For cell cycle analysis, the ethanol-fixed, RNAse (10 mg/mL, Sigma–Aldrich)-treated cells were stained PI (propidium iodide, 1 mg/mL, Sigma–Aldrich). For apoptosis analysis, cultured cells were stained with PI and and FITC-labeled Annexin V (BD, Franklin Lakes, NJ, USA). The cells were analyzed with Caliber (BD) and the data were further analyzed with Flowjo software (V10, BD). The statistical significance was measured by Student’s t-test.
7
Cell Proliferation and Apoptosis Assays
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A Cell-Light EdU DNA Cell Proliferation Kit (KeyGEN BioTECH, China) was carried out to evaluate cell proliferation. Brie y, cells were grown into 96-well plates. Then, cells were treated with 50 μmol/L EdU for 2 hours. Then, cells were xed using 4 % paraformaldehyde and then 1×Apollo reaction cocktail was used. Cell nuclei was stained using DAPI for 15 min. We captured the images using a uorescence microscope (Nikon, Tokyo, Japan).
Apoptosis assays PI and FITC-labeled annexin V (BD Biosciences, New Jersey, USA) staining was used to assess cell apoptosis. Cells were washed twice using PBS and resuspended using 100 μL 1× binding buffer, and then incubated with 5μL FITC-annexin V and PI with no light. Then, 400 μL 1× binding buffer was used. Within 1 hour, cells were exposed to ow cytometry analysis.
Apoptosis assays PI and FITC-labeled annexin V (BD Biosciences, New Jersey, USA) staining was used to assess cell apoptosis. Cells were washed twice using PBS and resuspended using 100 μL 1× binding buffer, and then incubated with 5μL FITC-annexin V and PI with no light. Then, 400 μL 1× binding buffer was used. Within 1 hour, cells were exposed to ow cytometry analysis.
8
Apoptosis Quantification by Flow Cytometry
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After the treatment, centrifugation at 1200 rpm for 3 min was performed to harvest the cells, with PBS washing twice. The harvested cells were resuspended in FITC-labeled Annexin V (5 μL; BD Bioscience, San Diego, CA, USA) as well as PI (5 μL; BD bioscience, CA, USA) in darkness for 5 min, with PBS washing three times. Finally, flow cytometry (FACSCalibur; BD bioscience, CA, USA) was adopted to calculate the cell apoptosis rate.
9
Metformin Induces Apoptosis and Cell Cycle Arrest
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Cell apoptosis detection kit (propidium iodide (PI), RNase staining buffer and FITC-labeled Annexin V) were purchased from BD Pharmingen (San Diego, CA, USA). Cells were seeded 2.5 × 105 per well in 6-well plates for 24 h. Then the medium was replaced by culture medium containing metformin 0, 20 or 40 mM for 24 or 48 h. The cells were harvested for analysis of cell cycle and apoptosis, respectively. The cell cycle was analyzed using PI staining, according to the manufacturer’s instructions. Briefly the cells were fixed in 70 % ethanol, stained with PI, and the amount of PI-labeled DNA in a cell was measured by a flow cytometer (Accuri C6, Becton Dickinson, San Jose, CA, USA). The acquired data were analyzed by FlowJo software (Ashland, OR, USA). To determine the apoptotic cells, the cells were stained with Annexin V-FITC and PI immediately after harvesting, and analyzed by flow cytometry, as described by the manufacturer’s instructions.
10
Cell Cycle and Apoptosis Analysis
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The cell cycle and phosphatidylserine (PS) externalization were detected by FCM. In short, the cell suspension was first filtered through 300-mesh nylon, and washed twice with cold PBS. Subsequently, the cells were suspended in PBS at a concentration of 1 × 10 6 cells/mL. For cell cycle assay, the cells were washed and resuspended in 500 mL propidium iodide (PI) (BD Biosciences, San Jose, CA, USA), and incubated in the dark for 30 min. For PS externalization assay, 5 mL FITC-labeled Annexin-V and 5 mL PI (BD Biosciences) were added into 500 mL of cell suspension, and incubated in the dark for 15 min. The stained hepatic cells were assayed with a FC500 MPL flow cytometer (Beckman Coulter, Fullerton, CA, USA).
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