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Propidium iodide

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Propidium iodide is a fluorescent dye that intercalates with DNA. It is commonly used in flow cytometry and cell biology research to assess cell viability and quantify DNA content.

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Lab products found in correlation

Pi rnase staining solution, by Cell Signaling Technology (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Cellquest version 3, by BD (3 mentions) Binding buffer, by BD (3 mentions) Annexin 5 fitc, by Cell Signaling Technology (3 mentions) Facsaria 2 cytometer, by BD (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Annexin 5 fluorescein isothiocyanate, by Cell Signaling Technology (2 mentions) Lsrfortessa x 20 flow cytometer, by BD (1 mentions) Anti γh2ax antibody, by Cell Signaling Technology (1 mentions) 5 bromo 2 deoxyuridine brdu, by Thermo Fisher Scientific (1 mentions) Macsquant vyb flow cytometer, by Miltenyi Biotec (1 mentions) Rnase a, by Thermo Fisher Scientific (1 mentions) Insulin, by Cell Signaling Technology (1 mentions) Dexamethasone (dex), by Cell Signaling Technology (1 mentions) Anti p ir, by Cell Signaling Technology (1 mentions) Horseradish peroxidase hrp conjugated polyclonal goat anti mouse and anti rabbit, by Santa Cruz Biotechnology (1 mentions) Anti glut1, by Cell Signaling Technology (1 mentions)

23 protocols using propidium iodide

1

Annexin V-FITC and PI Apoptosis Assay

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Following 48 h of incubation, 3×103 cells/well were seeded into 96-well plates and labeled with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; Cell Signaling Technology, Inc.), as per as the manufacturer's protocol. The BD LSRFortessa X-20 flow cytometer (BD Biosciences) with FlowJo software (version 10.0.6; BD Biosciences) was used to analyze cell apoptosis.
Ming M., Ying M, & Ling M. (2019). miRNA-125a-5p inhibits hepatocellular carcinoma cell proliferation and induces apoptosis by targeting TP53 regulated inhibitor of apoptosis 1 and Bcl-2-like-2 protein. Experimental and Therapeutic Medicine, 18(2), 1196-1202.
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2

Multiparametric Cell Cycle Analysis

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For cell cycle analysis, Huh7 and SNU-475 cells were pulsed with 30 μg/mL 5-Bromo-2´-Deoxyuridine (BrdU, Thermo Fisher Scientific) for 1 hour at 37°C. Cells were then trypsinized and fixed with 70% ethanol, permeabilized with 0.3% Triton-X 100 and incubated with a BrdU-FITC conjugated antibody (Thermo Fisher Scientific). Cells were stained with 50 ng/mL propidium iodide (Thermo Fisher Scientific), 10 μg/mL RNase A (Thermo Fisher Scientific), and incubated for 30 minutes at 37°C. For γH2AX analysis, cells were fixed in 70% ethanol, permeabilized with 0.3% Triton-X100 and blocked in 0.8% BSA. Cells were then incubated with an anti-γH2A.X antibody (#9718; Cell Signaling Technology), which was detected using a mouse-FITC IgG. Cells were also stained with propidium iodide as described above to identify 2N and >2N populations. For each sample, at least 40,000 cells were analyzed using the MACSQuant VYB Flow cytometer (MACS Miltenyi Biotec, Auburn, CA). All data were analyzed by FlowJo 10.0 software.
Kwan S.Y., Sheel A., Song C.Q., Zhang X.O., Jiang T., Dang H., Cao Y., Ozata D.M., Mou H., Yin H., Weng Z., Wang X.W, & Xue W. (2019). Depletion of TRRAP induces p53-independent senescence in liver cancer by downregulating mitotic genes. Hepatology (Baltimore, Md.), 71(1), 275-290.
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3

Investigating Cytokine Signaling in Cell Lines

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CAA was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, U.S.A.) to a concentration of 200 μM and stored at −20°C as a stock solution. The stock solution was diluted with Dulbecco’s modified Eagle’s medium (DMEM, HyClone, U.S.A) to a final concentration of 50 μM before use. Dexamethasone (Cell Signaling Technology, #9668) was diluted with DMEM to a final concentration of 1 μM. DMSO was added in the vehicle control group. TGF-β (240-B; R&D Systems, MN, U.S.A.) was diluted to a concentration of 6.4 ng/ml, and IL-6 (206-IL/CF, R&D Systems, MN, U.S.A.) was diluted to a concentration of 8 ng/ml before use. Insulin (Cell Signaling Technology, #9668), human IL-6 high sensitivity enzyme-linked immunosorbent assay (ELISA) Kit (Abcam, ab46042, U.S.A), human IL-17 High Sensitivity ELISA Kit (Abcam, ab46042, U.S.A), propidium iodide (Cell Signaling Technology, #9668), 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, #9668), primary antibodies including anti-IL-17RC, anti-IL-6, anti-p-IR (Cell Signaling Technology, #9542), anti-GLUT1 (Cell Signaling Technology, #9542), and monoclonal mouse anti-GAPDH (Santa Cruz, CA, U.S.A.), and horseradish peroxidase (HRP)–conjugated polyclonal goat anti-mouse and anti-rabbit (both from Santa Cruz, CA, U.S.A.) were used in the present study. Human IL-17RC adenovirus was purchased from Vector Biolabs (Malvern, PA, U.S.A.).
Gong Y., Liu H, & Tao L. (2019). Cajanonic acid A regulates the ratio of Th17/Treg via inhibition of expression of IL-6 and TGF-β in insulin-resistant HepG2 cells. Bioscience Reports, 39(12), BSR20181716.
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4

Cell Cycle Analysis of miR-34a Cells

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The miR-34a stable cells (3×105) were seeded in six-well plates. Cell samples were then stained with propidium iodide (Cell Signaling Technology, Inc.) at room temperature for 30 min. Cell cycle distribution analysis was assessed using an Accuri C5 flow cytometer (BD Biosciences) and quantified using the CellQuest Pro software (BD Biosciences).
Hwang T.I., Cuiu Y.C., Chen Y.C., Chen P.C., Tsai T.F., Chou K.Y., Ho C.Y., Chen H.E., Chang P.H, & Chang A.C. (2023). Tumor suppressive functions of hsa-miR-34a on cell cycle, migration and protective autophagy in bladder cancer. International Journal of Oncology, 62(5), 66.
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5

Cell Cycle Synchronization and Analysis

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For double thymidine block cells were seeded and 2mM thymidine was added and then later released from this block by washing the cells three times with PBS and adding complete media followed by second thymidine block and release. For synchronization using serum starvation and release, cells were kept in serum free media for 48hrs. For synchronizing cells in G1/S phase, cells were released for 2 hours and for synchronizing cells in G2/M phase, cells were released for 8 hours followed by drug treatment. In all cases the expected cell cycle was validated using FACS. For cell cycle analysis cells were fixed in cold ethanol and re-suspended in propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology). After incubation for 15 minutes at room temperature in the dark, flow cytometric analysis was performed on a FACS AriaII cytometer (BD Biosciences). Flow cytometry data was analyzed by using FlowJo software to measure polyploidy (>4N).
Shah K.N., Bhatt R., Rotow J., Rohrberg J., Olivas V., Wang V.E., Hemmati G., Martins M.M., Maynard A., Kuhn J., Galeas J., Donnella H.J., Kaushik S., Ku A., Dumont S., Krings G., Haringsma H.J., Robillard L., Simmons A.D., Harding T.C., McCormick F., Goga A., Blakely C.M., Bivona T.G, & Bandyopadhyay S. (2018). Aurora kinase A drives the evolution of resistance to third generation EGFR inhibitors in lung cancer. Nature medicine, 25(1), 111-118.
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6

Lapatinib Inhibits Breast Cancer Cell Viability

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Cells HCC1954, MCF-7, or MCF-7-HER2) were plated at a known density as described previously and then subsequently treated every day with vehicle (DMSO) or 2μM lapatinib up to 4 days. Total numbers of viable cells were counted using the Countess Cell Counter. Cell cycle analysis was conducted using propidium iodide according to the manufacturer’s instructions (Cell Signaling Technology).
Shah D., Wyatt D., Baker A.T., Simms P., Peiffer D.S., Fernandez M., Rakha E., Green A., Filipovic A., Miele L, & Osipo C. (2018). Inhibition of HER2 Increases Jagged1-dependent Breast Cancer Stem Cells: Role for Membrane Jagged1. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(18), 4566-4578.
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7

Apoptosis Analysis by TUNEL Assay

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Apoptosis was evaluated using the Dead-EndTM Fluorometric TUNEL System (Promega, Madison, WI, USA). Cells were fixed with 2% paraformaldehyde and permeabilized with 66% ice-cold ethanol for 2 h at 4 °C. Subsequently, the cells were rinsed with PBS and incubated in equilibration buffer for 5 min at RT. Fixed cells were incubated with fluorescein-12-dUTP in a reaction catalyzed by recombinant terminal deoxynucleotidyl transferase (TdT) for 1 h at 37 °C. After washing, cells were incubated with propidium iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Danvers, MA, USA) for 30 min at RT in darkness. Stained cells were analyzed by flow cytometry using a Cytomics FC 500 MPL system and quantified with MXP software (both from Beckman Coulter Brea, CA, USA).
García-Mato Á., Cervantes B., Rodríguez-de la Rosa L, & Varela-Nieto I. (2023). IGF-1 Controls Metabolic Homeostasis and Survival in HEI-OC1 Auditory Cells through AKT and mTOR Signaling. Antioxidants, 12(2), 233.
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8

Visualizing Apoptotic Cell Phagocytosis

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Murine apoptotic thymocytes were induced by exposure to UV irradiation at 312 nm for 10 min. Transfected J774.1 cells treated with IFNγ and LPS and apoptotic cells stained with propidium iodide (Cell Signaling Technology) were co‐cultured (1:10) for 60 min and then extensively washed with phosphate‐buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, and blocked with 3% bovine serum albumin in PBS. Actin was stained with Alexa Flour 488‐conjugated phalloidin (Invitrogen Life Technologies). The coverslips were mounted using PermaFluor Mounting medium (Thermo Fisher Scientific, Miami, FL, USA). For confocal immunofluorescence analysis, cells were visualized with a Zeiss LSM 700 confocal microscope using Zen software.
Atomura R., Sanui T., Fukuda T., Tanaka U., Toyoda K., Taketomi T., Yamamichi K., Akiyama H, & Nishimura F. (2016). Inhibition of Sprouty2 polarizes macrophages toward an M2 phenotype by stimulation with interferon γ and Porphyromonas gingivalis lipopolysaccharide. Immunity, Inflammation and Disease, 4(1), 98-110.
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9

Tumor-T Cell Cytotoxicity Assay

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Tumor cells were labeled with eFluor 670 fluorescent dye (Invitrogen) following the manufacturer’s protocol, then co-cultured with T cells at different effector:target (E:T) ratios in a 96-well flat-bottom tissue culture plate (Eppendorf). Wells containing tumor cells alone were used as a negative control. Cells were co-cultured in 250 μL complete RPMI-1640 media + IL-2 (2 ng/mL) for 16 or 40 h, depending on the target cell. Soluble recombinant BAFF (5 or 100 ng/mL) was also added in specified experiments. After co-culture, cells were transferred to a 96-well round-bottom plate and centrifuged in order to collect co-culture supernatant for cytokine production analysis. Cells were washed with PBS, then stained with propidium iodide (Cell Signaling) for 20 min at room temperature. Cells were then analyzed using flow cytometry. Tumor cell death was measured by gating on labeled tumor cells in the APC channel and recording the percentage of PI + cells, then subtracting the percentage of PI + cells in the tumor cell-only negative control.
Wong D.P., Roy N.K., Zhang K., Anukanth A., Asthana A., Shirkey-Son N.J., Dunmire S., Jones B.J., Lahr W.S., Webber B.R., Moriarity B.S., Caimi P, & Parameswaran R. (2022). A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers. Nature Communications, 13, 217.
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10

Cell Cycle Synchronization and Analysis

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For double thymidine block cells were seeded and 2mM thymidine was added and then later released from this block by washing the cells three times with PBS and adding complete media followed by second thymidine block and release. For synchronization using serum starvation and release, cells were kept in serum free media for 48hrs. For synchronizing cells in G1/S phase, cells were released for 2 hours and for synchronizing cells in G2/M phase, cells were released for 8 hours followed by drug treatment. In all cases the expected cell cycle was validated using FACS. For cell cycle analysis cells were fixed in cold ethanol and re-suspended in propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology). After incubation for 15 minutes at room temperature in the dark, flow cytometric analysis was performed on a FACS AriaII cytometer (BD Biosciences). Flow cytometry data was analyzed by using FlowJo software to measure polyploidy (>4N).
Shah K.N., Bhatt R., Rotow J., Rohrberg J., Olivas V., Wang V.E., Hemmati G., Martins M.M., Maynard A., Kuhn J., Galeas J., Donnella H.J., Kaushik S., Ku A., Dumont S., Krings G., Haringsma H.J., Robillard L., Simmons A.D., Harding T.C., McCormick F., Goga A., Blakely C.M., Bivona T.G, & Bandyopadhyay S. (2018). Aurora kinase A drives the evolution of resistance to third generation EGFR inhibitors in lung cancer. Nature medicine, 25(1), 111-118.
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