The cell cycle progression was determined by flow cytometer analysis [29] (link). Neuroblastoma cells were treated with SsnB or DMSO in DMEM with 10% FBS. Cells were collected after trypsinization with trypsin-EDTA solution (Atlanta Biologicals) and washed with 1× phosphate buffered saline (PBS). Approximately 1×106 cells were suspended in 300 µl of ice-cold 1× PBS, fixed by adding 700 µl of chilled absolute ethanol (to make final ethanol concentration 70%) drop wise and incubated for overnight at −20°C. Cells were pelleted at 1000× rpm for 5 min at 4°C, washed with 1× PBS for 3 times and stained with propidium iodide (Sigma-Aldrich, St. Louis, MO) by adding 1 ml of propidium iodide staining solution (containing 20 µg/ml propidium iodide and 10 µg/ml DNase-free RNase A in 0.1% Triton X-100/PBS) for 1 h on ice in dark. Samples were subjected to fluorescence-activated cell sorting analyses utilizing Beckman Coulter flow cytometer (Beckman Coulter, Indianapolis, IN). A minimum of 10,000 events were analyzed in each experiment.
Propidium iodide
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, Italy, Japan, France, Sao Tome and Principe, Macao, Canada, Spain, India, Belgium, Australia, Israel, Switzerland, Poland, Ireland, Argentina, Austria, Brazil, Sweden, Portugal, New Zealand, Netherlands, Slovakia, Norway, Hungary, Czechia, Denmark
Propidium iodide is a fluorescent dye commonly used in molecular biology and flow cytometry applications. It binds to DNA and is used to stain cell nuclei, allowing for the identification and quantification of cells in various stages of the cell cycle.
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Lab products found in correlation
Rnase a, by Merck Group (744 mentions)
Facscalibur, by BD (522 mentions)
Facscalibur flow cytometer, by BD (320 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (292 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (243 mentions)
Triton x 100, by Merck Group (235 mentions)
Hoechst 33342, by Merck Group (233 mentions)
Facscanto 2, by BD (185 mentions)
Rnase a, by Thermo Fisher Scientific (170 mentions)
Cellquest software, by BD (124 mentions)
Hoechst 33342, by Thermo Fisher Scientific (117 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (112 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (111 mentions)
Bovine serum albumin (bsa), by Merck Group (111 mentions)
Annexin 5 fitc, by BD (99 mentions)
Facscan, by BD (99 mentions)
Facsdiva software, by BD (96 mentions)
Accuri c6, by BD (94 mentions)
Calcein am, by Merck Group (90 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (89 mentions)
7 211 protocols using propidium iodide
1
Cell Cycle Analysis of Neuroblastoma Cells
2
Cell Cycle Analysis by Flow Cytometry
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For cell cycle analysis, nuclear DNA was stained with propidium iodide (Sigma-Aldrich; product no. P4170) followed by flow cytometric determination of the DNA content of single cells, as described previously [35 (link),40 (link)]. Briefly, cultured adherent cells were detached from 60 mm dishes with 0.05% trypsin-EDTA, washed with PBS, and fixed in ice-cold 70% ethanol (Daejung Chemical & Metals; product no. 4023-4110). After washing cells twice with PBS, cells were incubated in 10 μg of ribonuclease A (Sigma-Aldrich; product no. R4642) plus 22.5 μg of propidium iodide per ml PBS for 15 min at room temperature under protection from light, washed with PBS, isolated into the single-cell population, and gated in propidium iodide area versus height using the CytoFLEX flow cytometer and CytExpert software. Quantitation of cell numbers in cell cycle phases was performed using Modfit LT 5.0 software (Verity Software House, Topsham, ME, USA).
3
Apoptosis Induction by WEE1 Inhibitor
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Cells were incubated at 0.1 × 106 cells/mL with AZD1775 (WEE1 inhibitor, Selleckchem) with or without QVD-Oph (pan-caspase inhibitor, Selleckchem, No. S7311) for 18, 24 or 72 h. After incubation, cells were washed with 1% BSA in PBS and stained with Annexin V-FITC (IQP-120F, IQ products, Groningen, The Netherlands) for 20 min on ice. Cells were washed with 1% BSA in PBS and stained with propidium iodide (Sigma Aldrich) to assess early apoptosis (Annexin V positive/propidium iodide negative) and late apoptosis (Annexin V positive/propidium iodide positive). Apoptosis was measured by flow cytometry (FACSCalibur, BD Biosciences). Data were analysed in Winlist 3D (Verity Software house, Topsham, ME, USA).
4
Capsaicin Survival Assay for Streptococci
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Survival in presence of capsaicin was tested by the live/dead assay using SYBR Green I (Invitrogen, Eugene, OR, USA) and propidium iodide (Sigma–Aldrich), two nucleic acid dyes differing in their ability to penetrate bacterial cells (Magi et al., 2015 (link)). Briefly, overnight-grown streptococci were suspended in 1 mL capsaicin-supplemented BHI broth [∼1 × 108 colony forming units (CFU)/mL] and incubated for 15, 30, or 60 min at 37°C in 5% CO2. After staining with 1 × SYBR Green I and 40 μg/mL propidium iodide, cells were incubated at room temperature for 25 min in the dark, harvested on GTBP filters (Ø = 0.2 μm, Millipore, Billerica, MA, USA), and examined under an epifluorescence microscope (Axioskop2, Zeiss, Milano, Italy).
5
Cell Cycle Analysis by Flow Cytometry
6
Assessing Tumor Cell Phenotypes
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To assess tumor cell phenotypes, we first performed tumor cell proliferation MTT assay. In brief, after transfection with Wrap53 shRNA or negative control shRNA, cells were seeded into 6-well plates at 2×103 cells per well and cultured up to 5 days. To each culture well, 20 μl MTT reagent (Genview, USA) was added and further cultured for 4 h. Optical density was measured using a Microplate reader (Tecan Infinite, Switzerland) at 490/570 mm. Cell proliferation was calculated.
To assess tumor cell apoptosis, we employed Annexin V-APC staining. Tumor cells were infected with Wrap53 shRNA or negative control shRNA for 5 days, after which 10 μl Annexin V-APC reagent (eBioscience, USA) was added and further incubated for 10 min. Cell apoptosis rate was analyzed using flow cytometry (Millipore, USA).
Cell cycle distribution was assessed using propidium iodide staining and flow cytometry. Specifically, cells were grown and infected with lentiviruses, then detached using trypsin and reseeded into 6-cm dishes and grown for 3 days. Next, the cells were resuspended using trypsin and stained with propidium iodide (Sigma, St. Louis, MO, USA) at 37°C for 30 min in the dark. Cell cycle distribution was then analyzed using a flow cytometer (Millipore, USA).
To assess tumor cell apoptosis, we employed Annexin V-APC staining. Tumor cells were infected with Wrap53 shRNA or negative control shRNA for 5 days, after which 10 μl Annexin V-APC reagent (eBioscience, USA) was added and further incubated for 10 min. Cell apoptosis rate was analyzed using flow cytometry (Millipore, USA).
Cell cycle distribution was assessed using propidium iodide staining and flow cytometry. Specifically, cells were grown and infected with lentiviruses, then detached using trypsin and reseeded into 6-cm dishes and grown for 3 days. Next, the cells were resuspended using trypsin and stained with propidium iodide (Sigma, St. Louis, MO, USA) at 37°C for 30 min in the dark. Cell cycle distribution was then analyzed using a flow cytometer (Millipore, USA).
7
Streptococcus Survival with Carvacrol
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Group A streptococci survival in presence of carvacrol was studied by the live/dead assay as described previously (Zandri et al., 2012 (link)) using SYBR Green I (Invitrogen, Eugene, OR, USA) and propidium iodide (Sigma–Aldrich), two nucleic acid dyes differing in their ability to penetrate bacterial cells: bacteria with intact cell membranes stain fluorescent green, whereas those with damaged membranes stain fluorescent red. Briefly, overnight grown streptococci were suspended in 1 mL carvacrol-supplemented BHIB (∼1 × 108 CFU/mL) and incubated for 1, 3, or 24 h at 37∘C in 5% CO2. After staining with 1 × SYBR Green I and 40 μg/mL propidium iodide, samples were incubated at room temperature for 25 min in the dark, harvested on GTBP filters (Ø = 0.2 μm, Millipore, Billerica, MA, USA), and examined under an epifluorescence microscope (Axioskop 2, Zeiss, Milano, Italy).
8
Fluorescent Microscopy for Apoptosis and Necrosis
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WIF-B9 cells were tested for both apoptotic and necrotic cell death by fluorescence microscopic observation of cells stained with Hoechst 33342 (Life Technologies, Courtaboeuf, France) and propidium iodide (Sigma-Aldrich, Saint Quentin Fallavier, France).
After each treatment, cells were stained with 10 μg/ml Hoechst 33342 and 10 µg/mL propidium iodide in the dark for 15 min at 37°C. Cells were then examined under fluorescence microscopy (Olympus BX60, France). Total population was always more than 400 cells. Cells with condensed and/or fragmented chromatin were counted as apoptotic and propidium iodide-stained cells were counted as necrotic cells.
After each treatment, cells were stained with 10 μg/ml Hoechst 33342 and 10 µg/mL propidium iodide in the dark for 15 min at 37°C. Cells were then examined under fluorescence microscopy (Olympus BX60, France). Total population was always more than 400 cells. Cells with condensed and/or fragmented chromatin were counted as apoptotic and propidium iodide-stained cells were counted as necrotic cells.
9
Cell Cycle Analysis via Flow Cytometry
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Cells were trypsinized and harvested, pelleted and suspended in 1-ml propidium iodide solution (0.05 mg/ml propidium iodide (Sigma, Cat. # P4864), 0.1% sodium citrate, 0.1% Triton X-100). The cells were then treated with 10-ml 0.2 mg/ml of ribonuclease A (Colbiochem, Cat. #556746) for 30 min at room temperature, filtered through 40-μm nylon mesh, and analyzed by flow cytometry for the DNA content.
10
Adenovirus Transduction Effects on Cell Death
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LLC1 cells were transduced with adenovector concentration of 1.5 × 105 ifu/μl in DMEM. After 24 hours, transduction was supplemented with DMEM 10% FBS.
Phosphatydylserine exposure was determined by flow cytometry after staining with Annexin V, Alexa Fluor 488 conjugate (Thermo Scientific, A13201), and propidium iodide (Sigma, cat. no. P4170). DNA content was determined by propidium iodide staining after cell permeabilization with 70% ethanol.
The clonogenic assay was performed by plating 2000 transduced cells in 10-cm dishes, and colony formation was quantified 12 days later.
Calreticulin exposure was determined by surface staining using rabbit anticalreticulin polyclonal antibody 1:200 (Novus Biologicals, NB300-545) and goat antirabbit conjugate Alexa Fluor 488, 1:500 (Thermo Scientific, A11008) costained with propidium iodide.
Vesicular ATP content [45] (link), [46] (link) was assessed by incubating cells with 5 μM quinacrine (Sigma, Q3252-25G) in Krebs-Ringer solution during 30 minutes and then costaining with propidium iodide.
Phosphatydylserine exposure was determined by flow cytometry after staining with Annexin V, Alexa Fluor 488 conjugate (Thermo Scientific, A13201), and propidium iodide (Sigma, cat. no. P4170). DNA content was determined by propidium iodide staining after cell permeabilization with 70% ethanol.
The clonogenic assay was performed by plating 2000 transduced cells in 10-cm dishes, and colony formation was quantified 12 days later.
Calreticulin exposure was determined by surface staining using rabbit anticalreticulin polyclonal antibody 1:200 (Novus Biologicals, NB300-545) and goat antirabbit conjugate Alexa Fluor 488, 1:500 (Thermo Scientific, A11008) costained with propidium iodide.
Vesicular ATP content [45] (link), [46] (link) was assessed by incubating cells with 5 μM quinacrine (Sigma, Q3252-25G) in Krebs-Ringer solution during 30 minutes and then costaining with propidium iodide.
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