Annexin 5 propidium iodide

Manufactured by Thermo Fisher Scientific

Sourced in United States

Annexin V/propidium iodide is a flow cytometry reagent used for the detection and quantification of apoptosis and cell death. Annexin V binds to phosphatidylserine, which is exposed on the cell surface during apoptosis. Propidium iodide is a DNA-binding dye that stains cells with compromised cell membranes, indicating late-stage apoptosis or necrosis. This combination of markers allows for the discrimination of viable, early apoptotic, and late apoptotic/necrotic cells.

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Lab products found in correlation

Hoechst 33258, by Thermo Fisher Scientific (7 mentions) Crystal violet, by Merck Group (4 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Flow cytometry, by BD (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Facscalibur, by BD (2 mentions) Trypsinized, by Thermo Fisher Scientific (1 mentions) Fcs express software, by De Novo Software (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) Guava easycyte flowcytometry system, by Merck Group (1 mentions) Anti mouse h 2kb fitc, by Thermo Fisher Scientific (1 mentions) Fcs express 3, by De Novo Software (1 mentions) Wallac victor 1420 multilabel counter, by PerkinElmer (1 mentions) Mts assay kit, by Promega (1 mentions) Facstar, by BD (1 mentions) Lsm5 confocal microscope, by Zeiss (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions) Pi apoptosis kit, by Thermo Fisher Scientific (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Nf κb transcription factor assay kit, by Cayman Chemical (1 mentions)

28 protocols using annexin 5 propidium iodide

Cell Viability and Apoptosis Assays

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MTT was utilized to detect cell viability according to the manufacturer’s instructions. Briefly, the cells were seeded in 96-well culture plates and incubated at 37°C with 5% CO₂ for 72 hours. For the colony formation assay, 5,000 cells were plated in a six-well plate for 12 days. Colonies were subjected to methanol/acetone (1:1) fixation and stained with crystal violet.
Apoptosis was analyzed by nuclear staining with Hoechst 33258 (Invitrogen, Carlsbad, CA, USA), and Annexin V/propidium iodide (Invitrogen) staining followed by flow cytometry. The percentage of Annexin V+ cells (indicated in the two right quadrants) was plotted.

Sun L., Yang S., Chi G, & Jin X. (2018). Hsp90 inhibitor NMS-E973 exerts the anticancer effect against glioblastoma via induction of PUMA-mediated apoptosis. OncoTargets and therapy, 11, 1583-1593.

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Apoptosis and Mitochondrial Potential Assay

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Cells were placed in 6-well multiplies, after being pretreated with CA for 3 h; cells were stimulated with LPS for 12 h. To analyze apoptosis, the cells were trypsinized (Gibco, Grand Island, NY), washed twice with PBS, and then stained with annexin V/propidium iodide (Invitrogen Inc., Carlsbad, CA), and flow cytometric analysis was performed according to the manufacturer's instructions (Becton Dickinson, San Jose, California).
The mitochondrial membrane potential (ΔΨm) was measured using the mitochondrial potential sensor 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetra-ethyl-benz-imidazolyl-carbocyanine iodide (JC-1, Jiancheng, Nanjing, China). The cells were collected and incubated with 4 μM JC-1 for 20 minutes at 37°C in the dark. Extracellular JC-1 was removed by washing cells twice in PBS; then, the amount of JC-1 retained by 20,000 cells per sample was analyzed on a flow cytometer using 488 nm excitation with 530 nm and 585 nm bandpass emission filters (Becton Dickinson, San Jose, California).

Liu M., Fang G., Yin S., Zhao X., Zhang C., Li J, & Liu Z. (2019). Caffeic Acid Prevented LPS-Induced Injury of Primary Bovine Mammary Epithelial Cells through Inhibiting NF-κB and MAPK Activation. Mediators of Inflammation, 2019, 1897820.

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Measuring Cell Growth and Apoptosis

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Cell growth was measured by MTS, and apoptosis was analyzed by nuclear staining with Hoechst 33258 (Invitrogen) and Annexin V/propidium iodide (Invitrogen) staining followed by flow cytometry as described [15 (link)]. For crystal violet assays, the same number of cells were treated for 48 hours in 12-well plates, and the attached cells were stained with crystal violet (Sigma, St. Louis, MO) [49 (link)]. Details are found in the supplementary materials.

Li X., Li M., Ruan H., Qiu W., Xu X., Zhang L, & Yu J. (2016). Co-targeting translation and proteasome rapidly kills colon cancer cells with mutant RAS/RAF via ER stress. Oncotarget, 8(6), 9280-9292.

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Quantifying Cell Apoptosis by Flow Cytometry

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Cell apoptosis was assessed by annexinV/propidium iodide (Invitrogen) and by the Apo-BrdU flow kit (BD Pharmingen). Cells were fixed and treated with terminal deoxynucleotidyl transferase to expose DNA fragments, which were labeled by Br-dUTP and stained with FITC-conjugated anti-BrdU antibody according to the instructions of the manufacturer. Cell samples were then analyzed by FACS to determine proliferating or apoptotic fractions.

Liu G., Sun Y., Ji P., Li X., Cogdell D., Yang D., Parker Kerrigan B.C., Shmulevich I., Chen K., Sood A.K., Xue F, & Zhang W. (2014). MiR-506 Suppresses Proliferation and Induces Senescence by Directly Targeting the CDK4/6-FOXM1 Axis in Ovarian Cancer. The Journal of pathology, 233(3), 308-318.

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MTT Assay and Cell Cycle Analysis

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The colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT, Sigma) assay was performed as previously described [11 (link)]. Briefly, cells in 96 well-plates were
incubated with 100 µl of MTT (0.5 mg/ml) in phenol red-free medium for 2
hours at 37°C, switched into 200 µl of dimethyl-sulfoxide, and
shaken for 5 min at room temperature before measuring absorbance at 540 nm. Cell
death was measured by counting trypan blue-stained cells using the
hemocytometer. Annexin V/propidium iodide (Invitrogen) was stained, as
previously described [30 (link)].
The cell-cycle profile was analyzed as previously described [11 (link)]. Briefly, cells were washed with
ice-cold 0.2% bovine serum albumin in phosphate-buffered saline, and
resus-pended in 250 mM sucrose/40 mM citrate buffer (pH 7.6) containing
0.5% dimethylsulfoxide. Nuclei were prepared, stained with propidium
iodide, and analyzed using the Guava EasyCyte flowcytometry system
(MilliporeSigma, Billerica, MA, USA) with a gate that selects single nuclei
within a normal size range. The cell-cycle parameters from 5,000 gated nuclei
were determined, and subsequent analysis was performed using FCS EXPRESS
software (De Novo Software, Los Angeles, CA, USA).

Hong S.K., Wu P.K, & Park J.I. (2017). A cellular threshold for active ERK1/2 levels determines Raf/MEK/ERK-mediated growth arrest versus death responses. Cellular signalling, 42, 11-20.

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Annexin V Apoptosis Assay by Flow Cytometry

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Cell apoptosis assay was performed using the Annexin V/propidium iodide (Invitrogen) staining kit according to the manufacturer's instructions. Cells were stained with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide for 30 min and were analysed by flow cytometry to determine the percentage of apoptotic cells.

Wu S.Y., Rupaimoole R., Shen F., Pradeep S., Pecot C.V., Ivan C., Nagaraja A.S., Gharpure K.M., Pham E., Hatakeyama H., McGuire M.H., Haemmerle M., Vidal-Anaya V., Olsen C., Rodriguez-Aguayo C., Filant J., Ehsanipour E.A., Herbrich S.M., Maiti S.N., Huang L., Kim J.H., Zhang X., Han H.D., Armaiz-Pena G.N., Seviour E.G., Tucker S., Zhang M., Yang D., Cooper L.J., Ali-Fehmi R., Bar-Eli M., Lee J.S., Ram P.T., Baggerly K.A., Lopez-Berestein G., Hung M.C, & Sood A.K. (2016). A miR-192-EGR1-HOXB9 regulatory network controls the angiogenic switch in cancer. Nature Communications, 7, 11169.

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Apoptosis Analysis of GBM Cells

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The apoptosis of GBM cells was analyzed by nuclear staining with Hoechst 33,258 (Invitrogen), or Annexin V/propidium iodide (Invitrogen) followed by flow cytometry as described previously.18 (link) For the nuclear Hoechst 33,258 staining, the treated GBM cells were stained with Hoechst 33,258 (3.7% formaldehyde, 0.5% Nonidet P-40, and 10 ug/mL Hoechst 33,258 (Invitrogen)), and the condensed chromatin and micronucleation was counted under microscopic visualization.

Zhao H., Chen G, & Liang H. (2019). Dual PI3K/mTOR Inhibitor, XL765, suppresses glioblastoma growth by inducing ER stress-dependent apoptosis. OncoTargets and therapy, 12, 5415-5424.

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Apoptosis Evaluation by Flow Cytometry

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Cell apoptosis was evaluated by flow cytometry. In brief, the cells were resuspended with phosphate-buffered saline and stained with Annexin V/propidium iodide at room temperature for 20 min (Invitrogen, Inc., Carlsbad, CA, USA). The resulting fluorescence was detected using flow cytometry.

Cen X., Huang Y., Lu Z., Shao W., Zhuo C., Bao C., Feng S., Wei C., Tang X., Cen L., Guo W., Tian X., Tang Q, & Huang X. (2021). LncRNA IGFL2-AS1 Promotes the Proliferation, Migration, and Invasion of Colon Cancer Cells and is Associated with Patient Prognosis. Cancer Management and Research, 13, 5957-5968.

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Quantifying MHC-I and Apoptosis in Lung Cancer

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MHC-I expression on human or murine lung cancer cells was determined using anti-human HLA-ABC-FITC (eBioscience, San Diego, CA, 11–9983) or anti-mouse H-2K^b-FITC (eBiosciences, 11-5958) antibody for 30 min at 4°C. Cells were then washed twice with phosphate buffered saline (PBS) and subjected to flow cytometry (Becton, Dickinson and Company, Franklin Lakes, NJ). The mouse IgG2a Kappa isotype control FITC antibody (eBioscience, 11–4724) was used as control.
Apoptotic cell death was determined by staining with Annexin V/propidium iodide (PI) (Invitrogen, V13241). Cells were harvested and washed once with PBS, then resuspended in 100 μl binding buffer followed by incubation with 2.5 μl Annexin V per test for 20 min. Then, 1 μl PI per test was added and stained cells were subjected to flow cytometry.

Yuan D., Xia M., Meng G., Xu C., Song Y, & Wei J. (2015). Anti-angiogenic efficacy of 5′-triphosphate siRNA combining VEGF silencing and RIG-I activation in NSCLCs. Oncotarget, 6(30), 29664-29674.

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Apoptosis and Colony Formation Assays

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Apoptosis was analyzed by nuclear staining with Hoechst 33258 (Invitrogen), and Annexin V/propidium iodide (PI) (Invitrogen) followed by flow cytometry as described (50 (link)). For colony formation assays, the same number of cells were treated and plated in 12-well plates at appropriate dilutions, and allowed to grow for 10–14 days before staining with crystal violet (Sigma, St. Louis, MO) (16 (link)).

He K., Zheng X., Li M., Zhang L, & Yu J. (2015). mTOR inhibitors induce apoptosis in colon cancer cells via CHOP-dependent DR5 induction upon 4E-BP1 dephosphorylation. Oncogene, 35(2), 148-157.

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