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Annexin 5 and propidium iodide

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Annexin V and propidium iodide are commonly used as a pair of fluorescent dyes for analyzing cell viability and apoptosis. Annexin V binds to phosphatidylserine, which is exposed on the cell surface during early apoptosis. Propidium iodide is a DNA-binding dye that enters cells with compromised membranes, such as late-stage apoptotic or necrotic cells. The combination of these two dyes enables the identification of viable, early apoptotic, and late apoptotic/necrotic cells.

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Lab products found in correlation

Cellquest software, by BD (1 mentions) Biacore, by GE Healthcare (1 mentions) High performance liquid chromatography (hplc), by Shimadzu (1 mentions) Propidium iodide solution, by Keygen Biotech (1 mentions) Facscalibur flow cytometer, by Keygen Biotech (1 mentions) Flow cytometry, by BD (1 mentions) Fluorescence activated cell sorting (facs), by BD (1 mentions)

5 protocols using annexin 5 and propidium iodide

1

Cell Cycle and Apoptosis Analysis

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Flow cytometry was used for cell cycle and apoptosis analyses at 48 h post-transfection. For the cell cycle analyses, the cells were collected by trypsinization and fixed with 75% ethanol at −20 °C for 24 h. Next, the cells were incubated with a propidium iodide/PBS staining buffer (KeyGen, Nanjing, CHN) at 37 °C for 30 min, after which the percentages of G1, S and G2/M phase cells were calculated using Cell Quest software. For the apoptosis assays, the cells were treated by Annexin-V and propidium iodide (KeyGen, Nanjing, CHN) and analyzed within 30 min after staining. Quantification of fluorescence was performed by flow cytometry.
Li B., Guo X., Li N., Chen Q., Shen J., Huang X., Huang G, & Wang F. (2020). WNT1, a target of miR-34a, promotes cervical squamous cell carcinoma proliferation and invasion by induction of an E-P cadherin switch via the WNT/β-catenin pathway. Cellular Oncology (Dordrecht), 43(3), 489-503.
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2

Cell Cycle and Apoptosis Analysis

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For DNA content analysis, cells were trypsinized, washed with PBS, and fixed in 75% ice‐cold ethanol at 4°C overnight. Cells were then rehydrated in PBS. Following RNase digestion, cells were stained with propidium iodide. Flow cytometry analysis was performed using red (propidium iodide) emission (at 630 nm). Data from 10,000 cells were collected and analyzed by using CellQuest software (Becton Dickinson). For apoptosis analysis, cells were double‐stained with annexin V and propidium iodide (KeyGen) and subjected to flow cytometric analysis.
Liu X., Tan Y., Zhang C., Zhang Y., Zhang L., Ren P., Deng H., Luo J., Ke Y, & Du X. (2016). NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2. EMBO Reports, 17(3), 349-366.
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3

Characterization of sDR5-Fc Fusion Protein

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sDR5-Fc was designed to contain the extracellular fragment of human DR5 (amino acids 1 to 182) and the human IgG1 Fc fragment. It was expressed in CHO-K1 cells by stable transfection and purified from culture supernatant using Protein A affinity column (53 (link)). The quality of the purified protein was assessed by SDS–polyacrylamide gel electrophoresis and high-performance liquid chromatography (Shimadzu). Endotoxins were measured using a Limulus amebocyte lysate assay (Xiamen Bioendo Technology Co. Ltd.). Flow cytometry was performed to examine the blocking effect of sDR5-Fc on TRAIL-induced Jurkat cell apoptosis after staining cells with annexin V and propidium iodide (KeyGen Biotech). The binding activity of sDR5-Fc with human and murine TRAIL was measured by Biacore (X100, GE Life Sciences), with TRAIL precoated on the bottom of the chip. Association rates (Ka) and dissociation rates (Kd) were acquired by a simple 1:1 binding model. The equilibrium dissociation constants (KD) was calculated as the ratio of Kd and Ka (KD = Kd/Ka).
Wang Y., Zhang H., Wang Z., Wei Y., Wang M., Liu M., Wang X., Jiang Y., Shi G., Zhao D., Yang Z., Ren Z., Li J., Zhang Z., Wang Z., Zhang B., Zong B., Lou X., Liu C., Wang Z., Zhang H., Tao N., Li X., Zhang X., Guo Y., Ye Y., Qi Y., Li H., Wang M., Guo R., Cheng G., Li S., Zhang J., Liu G., Chai L., Lou Q., Li X., Cui X., Gao E., Dong Z., Hu Y., Chen Y.H, & Ma Y. (2020). Blocking the death checkpoint protein TRAIL improves cardiac function after myocardial infarction in monkeys, pigs, and rats. Science translational medicine, 12(540), eaaw3172.
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4

Cell Cycle and Apoptosis Assay Protocol

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For cell cycle assays, transfected cells were harvested, diluted to a density of 1 × 106 cells/mL and fixed with 70% ice-cold ethanol. Next, the cells were stained with 400 μL of a propidium iodide solution (Keygen Biotech, Nanjing, China) for 30 min and then subjected to cell cycle analysis using flow cytometry (BD Biosciences, San Jose, CA, USA).
For apoptosis analysis, 300 nM H2O2 was applied to induce apoptosis. Cells were collected 48 h after transfection and resuspended in binding buffer. The cells were then incubated with annexin V and propidium iodide (Keygen Biotech, Nanjing, China) for 15 min in the dark and then analyzed by flow cytometry on a FACSCalibur flow cytometer.
Xu Y., Yu J., Huang Z., Fu B., Tao Y., Qi X., Mou Y., Hu Y., Wang Y., Cao Y., Jiang D., Xie J., Xu Y., Zhao J, & Xiong W. (2020). Circular RNA hsa_circ_0000326 acts as a miR-338-3p sponge to facilitate lung adenocarcinoma progression. Journal of Experimental & Clinical Cancer Research : CR, 39, 57.
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5

Apoptosis and Cell Cycle Analysis

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Quantification of apoptosis induced by CDDP or CDDP combined with Cdc6 RNAi was performed with Annexin V and Propidium Iodide (PI) staining according to the manufacturer's (KeyGEN). Briefly, 1×105 cells were resuspend in Annexin V binding buffer and stained with Annexin V-FITC and Propidium Iodide (1μg/ml). After incubation at room temperature for 15 min, the apoptotic cell was quantified by flow cytometry.
For cell cycle analysis, cells were fixed in 70% ethanol overnight at 4°C. Fixed cells were then washed once in ice-cold PBS and stained with propidium iodide (PI) staining solution (50μg/ml PI, 100μg/ml RNase, 0.05% Triton X-100 in PBS) for 30 min. PI-stained cells were then analyzed for their DNA content by using FACS (BD Biosciences, San Jose, CA, USA).
Chen S., Chen X., Xie G., He Y., Yan D., Zheng D., Li S., Fu X., Li Y., Pang X., Hu Z., Li H., Tan W, & Li J. (2016). Cdc6 contributes to cisplatin-resistance by activation of ATR-Chk1 pathway in bladder cancer cells. Oncotarget, 7(26), 40362-40376.
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