Trout peripheral bloods were collected with a needle containing 0.1% heparin solution (FUJIFILM Wako Pure Chemical Corporation). After fixation with chilled 70% ethanol at 4 °C, blood samples were centrifuged at 1000 rpm at 4 °C for 10 min. The collected blood-cell pellets were stained with propidium iodide solution (100 µg/ml; Dojindo) containing 35 µg/ml RNase A (Sigma-Aldrich) for 16 h at 4 °C. The fluorescent intensity of the stained cells was determined using flow cytometry (Guava easyCyte).
Propidium iodide solution
Manufactured by Dojindo Laboratories
Sourced in Japan
Propidium iodide (PI) solution is a fluorescent dye used in cell biology applications. It binds to nucleic acids, specifically DNA, and emits red fluorescence upon excitation. This property makes PI a useful tool for identifying and quantifying dead or membrane-compromised cells in cell populations.
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Lab products found in correlation
Rnase a, by Thermo Fisher Scientific (4 mentions)
Microplate spectrophotometer, by Bio-Rad (2 mentions)
Annexin 5 fitc, by Dojindo Laboratories (2 mentions)
Annexin 5 binding solution, by Dojindo Laboratories (2 mentions)
Cck 8 kit, by Dojindo Laboratories (2 mentions)
Rnase a, by Merck Group (1 mentions)
Propidium iodide, by Olympus (1 mentions)
Fv3000, by Olympus (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Macs separation beads, by Miltenyi Biotec (1 mentions)
Facsaria 3, by BD (1 mentions)
Macsquant flow cytometer, by Miltenyi Biotec (1 mentions)
Rnase a, by Dojindo Laboratories (1 mentions)
10 protocols using propidium iodide solution
1
Trout Blood Cell Analysis via Flow Cytometry
2
Embryo Imaging with Propidium Iodide
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Mature seeds were cut in half through the center of the embryo, and pistils containing embryos at 3–10 DAP were fixed in a 1:1:18 solution of 37% formaldehyde, acetic acid and ethanol under vacuum two times for 1 h each and then overnight at 4℃. After gradually replacing ethanol with 1× PBS buffer, samples were stained with 5.0 µg/ml Propidium iodide solution (Dojindo, Kumamoto, Japan) containing 10.0 µg/ml RNase A (Invitrogen) in 1× PBS buffer overnight in the dark at 4℃. After dehydration in a graded ethanol series, ethanol was replaced with methyl salicylate for transparency. Propidium iodide fluorescence emission at 570–670 nm (excitation at 559 nm) was captured using a confocal laser scanning microscope (FV3000, Olympus, Shinjyuku, Tokyo, Japan).
3
Apoptosis Quantification in Transfected HL-60 Cells
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Following plasmid transfection for 48 h, the transfected HL-60 cells (1x106) were washed with PBS and resuspended in 500 µl binding buffer. Afterwards, the solution was transferred to a flow cytometry tube and mixed with 5 µl Annexin V-fluorescein isothiocyanate staining solution (BD Biosciences). Cells were treated with 10 µl propidium iodide solution (50 µg/ml; Dojindo Laboratories, Inc.) for 30 min at room temperature in the dark. The percentages of apoptotic cells were quantitated using a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (version 7.0; Tree Star, Inc.). The apoptosis rate was determined by calculating the percentage of early and late apoptotic cells.
4
Cell Cycle Analysis by Flow Cytometry
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For cell cycle analysis by flow cytometry, 5×104 DLD-1 cells were transfected with each miR in a 24-well plate, trypsinized after 72 h, washed with phosphate-buffered saline (PBS), and fixed in 70% ethanol on ice. After centrifugation, cells were stained with 50 mg/ml propidium iodide (PI) solution (Dojindo Molecular Technologies) and 0.1 mg/ml RNase A (Invitrogen) and analyzed by flow cytometry using a FACS BD FACSAria III cell sorter. Each histogram was constructed with data from at least 10,000 events and was used to calculate the percentage of the cell population in each phase.
5
Evaluating Cell Death and HER2 Expression
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Cell death assays were performed by using the Propidium Iodide (PI) solution (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, following drug treatment, cells were harvested and stained with PI solution in dark for 15 min. For flow cytometric analysis of HER2 protein levels present on the cell surface, cells were stained with APC anti-HER2 antibody (324407, Biolegend, CA, USA) or APC mouse IgG1 Isotype control antibody (400119, Biolegend). Stained cells were analyzed on NovoCyte Fluidics Station II (Agilent, CA, USA).
6
Cell Proliferation and Apoptosis Assays
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Cell proliferation was evaluated at indicated time points using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer’s protocol. CCK-8 reagent (10%) was added to each well for 3 h at 37 °C. Viability was evaluated by measuring the absorbance at a 450-nm wavelength with using a microplate spectrophotometer (BioRad).
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 1000 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. The apoptosis assays were run and analyzed with BD Jazz.
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 1000 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. The apoptosis assays were run and analyzed with BD Jazz.
7
Cell Cycle Analysis with Ubenimex
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For cell cycle analysis, each type of cells were incubated with different doses of ubenimex for 16 h, and then fixed in 70% ethanol on ice. After centrifugation, cells were stained with 50 mg/ml propidium iodide (PI) solution (Dojindo Molecular Technologies, kumamoto, Japan) and 0.1 mg/ml RNase A (Invitrogen). Stained cells were analyzed using flow cytometry on a FACSAria II. Histogram was constructed with data from at least 20,000 events. Besides, in the G1 phase of the cell cycle, geminin was degraded. Therefore, only cdt1 tagged with RFP was present and appeared as red fluorescence within the nuclei. In the S, G2, and M phases, cdt1 was degraded and only geminin tagged with GFP remained, resulting in cells with green fluorescent nuclei. Flow cytometric analyses were performed using FlowJo software.
8
Cell Cycle Analysis with Anti-cancer Drugs
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For cell cycle analysis, cells were incubated with each anticancer drug, alone or with ubenimex (100 μg/ml) for 48 h, then fixed in 70% ethanol on ice. After centrifugation, cells were stained with 50 mg/ml propidium iodide (PI) solution (Dojindo Molecular Technologies, Kumamoto, Japan) and 0.1 mg/ml RNase A (Invitrogen). Stained cells were analyzed with flow cytometry on a FACSAria II. Each histogram was constructed with data from at least 20,000 events. Flow cytometric analyses were performed with the FlowJo software (Digital Biology, Tokyo, Japan).
9
Isolation and Characterization of Murine Immune Cells
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Cell suspensions from the lymph nodes or the spleens of 6- to 12-week-old mice were stained with specific antibodies (listed in the ‘Reagents’ section) and sorted using a FACS Aria III (BD Biosciences, San Jose, CA, USA) with a typical final purity of >97%. Where required, CD8+ cells and APCs were sorted using magnetic MACS Separation Beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. For flow cytometry, cells were stained with specific antibodies for 30 min on ice after blocking Fc receptors with anti-CD16/CD32. Propidium iodide (PI) solution (Dojindo Laboratories, Kumamoto, Japan) was added at 0.1 µg ml−1 to exclude dead cells. Stained cells were examined using a MACS Quant flow cytometer (Miltenyi Biotec). Live cells were identified as PI− cells with appropriate intensities on FSC and SSC, and further gating was performed as described in the figure legends using FlowJo software (FlowJo LLC, Ashland, OR, USA).
10
Cell Proliferation and Apoptosis Assays
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Cell proliferation was determined at indicated time points using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer's protocol. We added 10% of CCK-8 solution to each well for 3 h before measuring the absorbance at 450 nm using a microplate spectrophotometer (Bio-Rad).
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 100 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. For cell cycle assays, cells were fixed in 70% precooled ethanol on ice for 2 h, centrifuged at 100 rpm for 5 min, and resuspended with 400 μl PI and 100 μl RNaseA (Dojindo). After a 30 min incubation at 4°C, cells were washed and resuspended with PBS. Both the apoptosis assays and cell cycle assays were run and analyzed with BD Jazz.
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 100 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. For cell cycle assays, cells were fixed in 70% precooled ethanol on ice for 2 h, centrifuged at 100 rpm for 5 min, and resuspended with 400 μl PI and 100 μl RNaseA (Dojindo). After a 30 min incubation at 4°C, cells were washed and resuspended with PBS. Both the apoptosis assays and cell cycle assays were run and analyzed with BD Jazz.
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