The human pancreatic cancer cell lines MiaPaPc-2, BxPC-3 and PANC-1 were purchased from the American Type Culture Collection. The S2VP10 cells, derived from the Suit-2 cells as reported30 (link), were originally provided by Dr. M. Hollingsworth (University of Nebraska). Cell line authentication was determined by short tandem repeat analysis (STR) performed by the American Type Culture Collection. OGT overexpression MiaPaPc-2 and BxPC-3 cells were generated by transfection of pCMV-flag-OGT (OGT, Addgene, #29760); and OGT knockdown S2VP10 and PANC-1 cells were generated by infected with lentiviruses containing a short hairpin RNA (shRNA) specific for OGT (shOGT, Addgene, #10878). PANC-1 cells with FADD knockdown were established as we previously described by shRNA targeting FADD (Open Biosystems)31 (link). All stable clones were selected by 2 μg/ml puromycin as we previously reported31 (link). Sequencing analysis was performed to confirm the correct sequence, and protein expression was validated by Western blot analysis. Apoptosis was induced by TRA-8, a DR5 agonist antibody that was generated as previously described32 (link); and determined by Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA) and analyzed by flow cytometry as we previously reported11 (link).
Annexin 5 fluoresce in isothiocyanate fitc and propidium iodide staining kit
Manufactured by BD
Sourced in United States
The Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit is a laboratory reagent used to detect and quantify apoptosis and cell viability. The kit contains Annexin-V-FITC, which binds to phosphatidylserine residues exposed on the cell surface during apoptosis, and propidium iodide, which stains the DNA of cells with compromised cell membranes. The kit enables the simultaneous detection of early apoptotic, late apoptotic, and necrotic cells using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Flow cytometry, by BD (2 mentions)
Panc 1, by Addgene (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Bxpc 3, by American Type Culture Collection (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscan instrument, by BD (1 mentions)
Interleukin 6 (il 6), by Merck Group (1 mentions)
8 protocols using annexin 5 fluoresce in isothiocyanate fitc and propidium iodide staining kit
1
Modulation of Pancreatic Cancer Cell Apoptosis
2
Annexin V-FITC and PI Apoptosis Assay
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Apoptosis was evaluated microscopically and by a flow cytometry-based assay using the annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San Diego, CA, USA). Apoptotic cells were indicated as annexin-V-positive. Cells were trypsinized, washed twice with PBS and centrifuged at 1000 rpm for 5 min at room temperature. Then, cells were resuspended in 1× binding buffer, 5 μL of PI and FITC were added per 1 × 105 cells, cells were vortexed, and incubated for 15 min at room temperature in dark. Subsequently, cells were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
3
Apoptosis Analysis via Flow Cytometry
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Apoptosis was analyzed using the AnnexinV‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA, USA) following the manufacturer's instruction. Samples were analyzed by using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
4
Apoptosis Analysis by Flow Cytometry
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The induction of apoptosis associated with changes in miR-92a expression was evaluated using a flow cytometry assay based on the annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San Diego, CA, USA). Annexin-V-positive cells were considered apoptotic. The transfected cells were trypsinized, washed twice with PBS, and centrifuged at 1000 rpm for 5 min at room temperature. Then, the cells were resuspended in 1 × binding buffer (0.01M Hepes (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl2), 5 µL of PI and FITC solutions were added per 1×105 cells, cells were vortexed, and incubated for 15 min at room temperature in the dark. The apoptotic rate of the cells was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).34 (link)
5
Annexin-V-FITC and PI Staining for Apoptosis
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Apoptosis was determined by flow cytometry based on the Annexin-V-fluoresce in isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San Diego, USA).54,55 (link) See ESI.†
6
Apoptosis Assessment of Compound 7f
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Flow cytometric analysis using Annexin-V-fluoresce in isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San Diego, USA) was performed to assess the apoptosis. At 70% confluence, the cells were treated with compound 7f at different concentrations of (0–8 μmol/l) for 24 h. Then after trypsinization of cells, washing with PBS and centrifugation, the cells were mixed with FITC and PI in binding buffer. Becton Dickinson (Franklin Lakes, NJ, USA) was used to do the flow cytometry analysis16 (link),17 (link).
7
Annexin-V-FITC and PI Apoptosis Assay
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Apoptosis was determined by flow cytometry based on the Annexin-V-fluoresce in isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San Diego, USA) [40 (link), 41 (link)] See Appendix A.
8
Assessing Apoptosis in U-2OS and MG63 Cells
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Cell apoptosis rate was assessed by Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Biosciences, Franklin Lakes, NJ, USA). U-2OS and MG63 cells were transduced with shRNAs and treated with or without 50 ng/mL IL-6 (Sigma-Aldrich Co.) for 24 h. At the end of culture period, both adherent and floating cells were harvested and then labeled with Annexin V-FITC and PI. Cell apoptosis was then analyzed using a FACScan instrument (BD Biosciences).
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