Single-cell suspensions of skin and dLN cells were incubated with anti-CD16/CD32 (TONBO Biosciences) for 5 min, and then stained using the fluorochrome-conjugated antibodies described in Table S1 . Intracellular GzmB and CTLA-4 were stained using Fixation Buffer and 10 × Permeabilization Wash Buffer (BioLegend) as described in the manufacturer's protocol. The fluorescence intensity of KikGR-Red was reduced by fixation, thus, the proportion of KikGR-Red+ cells was lower in samples subjected to intracellular staining than in fresh samples. Dead cells were stained with 7-AAD (eBioscience) or propidium iodide (Wako) and excluded from our analyses. Stained samples were analyzed using a SP6800 Spectral Cell Analyzer (SONY). All data were exported as FCS files and analyzed using FlowJo software (Tree Star).
Propidium iodide
Manufactured by Fujifilm
Sourced in Japan
Propidium iodide is a fluorescent dye used in various laboratory applications. It is a nucleic acid stain that binds to DNA, emitting red fluorescence when excited by a specific wavelength of light. Propidium iodide is commonly used in flow cytometry and fluorescence microscopy to identify and quantify cells.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Fujifilm (7 mentions)
Triton x 100, by Fujifilm (4 mentions)
Propidium iodide, by Thermo Fisher Scientific (4 mentions)
Rnase a, by Thermo Fisher Scientific (4 mentions)
Rnase a, by Nacalai Tesque (3 mentions)
Rnase a, by Fujifilm (3 mentions)
Rnase, by Fujifilm (2 mentions)
The tail image based cytometer, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Merck Group (2 mentions)
Rnase a, by Qiagen (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Phosphate buffered saline (pbs), by Fujifilm (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Fixation buffer, by BioLegend (1 mentions)
Permeabilization wash buffer, by BioLegend (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Sp6800 spectral cell analyzer, by Sony (1 mentions)
Facsverse, by BD (1 mentions)
Streptomycin sulfate, by Fujifilm (1 mentions)
50 protocols using propidium iodide
1
Multiparametric Flow Cytometry of Immune Cells
2
Measuring Plant Root Cell Length and Meristem
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To obtain fully elongated cortical cell length measurements and meristematic cell counts, the root tips of plants grown on solid media were stained and observed. Under severely low B (0.1 μM), primary roots (PR) of 4-d-old plants were used, in which Col-0 had shorter roots than the tmn1 mutants, but in which root tip cells had not severely collapsed. PRs were collected directly from solid media and stained with 10 μg ml–1 propidium iodide (PI; FUJIFILM Wako Pure Chemical, Japan) for 15 min, then rinsed twice in ultrapure water. Under normal B (100 μM), 11-d-old plants were used, in which root growth inhibition was obvious in the mutants. PRs were collected directly from solid media and stained with PI as described above for 40 min. Images were obtained using a confocal laser scanning microscope (LSM510, Zeiss, Germany). The excitation and detection wavelength windows were set at 488 and >540 nm, respectively. Up to three fully elongated cortical cells were selected from each PR; their longitudinal lengths were measured using ImageJ software (https://imagej.nih.gov/ij/ ). The numbers of cortical cells between the quiescent centre and the first elongating cortical cell were counted in the PI-stained PRs. Counting was performed using the Cell Counter ImageJ plugin (https://imagej.nih.gov/ij/plugins/cell-counter.html ).
3
Cell Viability Assessment via Flow Cytometry
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Strain was precultured in liquid YPD medium at 25°C, and a stationary phase culture was diluted 100 folds to 10 ml of a new YPD. Cell suspension was diluted 10 folds to normal saline buffer containing 10 μg/ml of propidium iodide (Wako Pure Chemical Industries, Japan) and 1 mg/ml of RNase (Wako Pure Chemical Industries, Japan) and incubated at 37°C for 2 h. Stained cells were diluted 10 folds to sterilized distilled water and applied to a flow cytometry, On-chip Sort (FISHMAN, On-Chip Biotechnologies, Japan) according to the procedure manuals.
4
Propidium Iodide Staining for Apoptosis
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Late apoptosis/necrosis was evaluated by staining for propidium iodide (Wako). In brief, A549 cells (A549‐shScramble and A549‐shTFF‐1‐A) (5 × 105 cells) were seeded on a 6‐cm dish; after 4 days, the cells were trypsinized and resuspended at 1 × 106 cells/100 μL in RPMI. Thereafter, propidium iodide at a concentration of 50 μg/mL was added and incubated at room temperature for 5 minutes. Subsequent analysis was carried out using FACSVerse (Becton Dickinson) and FlowJo version 10.6.1 software. Results are calculated as mean ± SD of values from 3 individual experiments.
5
Photosensitizer-mediated Cell Imaging
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All reagents and solvents purchased were the highest commercial quality and used without further purification otherwise noted. Dimethyl sulfoxide (DMSO) was purchased from KANTO chemicals. Propidium iodide (PI), benzylpenicillin potassium, and streptomycin sulfate were purchased from Fujifilm Wako Chemicals. 1,3-Diphenylisobenzofuran (DPBF) was purchased from Sigma-Aldrich. Minimum essential medium (MEM) and phosphate-buffered saline (PBS) were purchased from Nacalai Tesque. Fetal bovine serum (FBS) was purchased from Capricorn Products Inc. The Ir complex 7 was synthesized according to our previous paper [26 (link)], a stock solution of which in DMSO was prepared and stored at 0 °C prior to use. UV-Vis absorption spectra of DPBF were measured on a JASCO V-550 UV-vis spectrophotometer. The microscopic images of HeLa S3 cells were observed on fluorescent microscopy (Biorevo, BZ-x800, Keyence, Amagasaki, Japan).
6
Cellular DNA Content Quantification
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Quantification of cellular DNA content at 72 h after transfection with control miRNA or #12 (5 nM) was determined by using a cytometer. Briefly, the cells were harvested and fixed with 70% cold ethanol at −20°C overnight. The fixed cells were then washed twice with PBS and resuspended in 100 μL PBS-based propidium iodide solution containing 0.1% Triton X-100 (Wako, Osaka, Japan), 0.2 mg/mL RNase A (Life Technologies), and 20 μg/mL propidium iodide (Life Technologies). Thereafter, they were incubated for 30 min at room temperature protected from the light. The DNA content in the cells was analyzed with a cytometer (The Tail Image-Based Cytometer, Life Technologies).
7
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested using trypsin/EDTA and fixed in 70% ethanol. Fixed cells were washed with PBS and resuspended in solution containing 20 μg/mL of propidium iodide (Fujifilm Wako), 200 μg/mL of RNase A (Nacalai Tesque), and 0.1% Triton X-100. Cells were analyzed by flow cytometry using SH800ZDP (Sony). Data were analyzed using FlowJo.
8
Cell DNA Content Quantification
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Quantification of cellular DNA content at 48 h after transfection with non-specific control, miR-92a (10, 20 or 40 nM) or siR-Dkk-3 (1, 2 or 5 nM) was determined by using a cytometer. Briefly, the cells were harvested and fixed with 70% cold ethanol at −20 °C overnight. The fixed cells were washed twice with PBS, resuspended in 100 μL PBS-based propidium iodide solution containing 0.1% Triton X-100 (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 0.2 mg/mL RNase A (Invitrogen) and 20 μg/mL propidium iodide (Invitrogen) and incubated for 30 min at room temperature protected from the light. The DNA content in the cells was analyzed through the cytometer (The Tali® Image-Based Cytometer, Invitrogen).
9
Cellular DNA Content Quantification
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Quantification of cellular DNA content at 48 h after treatment with CRC-EVs was determined by using a cytometer. Briefly, the cells were harvested and fixed with 70% cold ethanol at -20°C overnight. The fixed cells were washed twice with PBS, resuspended in 100 μl PBS-based propidium iodide solution containing 0.1% Triton X-100 (Wako, Osaka, Japan), 0.2 mg/ml RNase A (Life Technologies), and 20 μg/ml propidium iodide (Life Technologies), and incubated for 30 min at room temperature protected from the light. The DNA content in the cells was analyzed through the cytometer (The Tail® Image-Based Cytometer, Life Technologies).
10
Cell Cycle Analysis by Flow Cytometry
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Cells were fixed with 70% (v/v) ethanol at -20°C overnight. They were then centrifuged at 5,000 r.p.m at 4°C for 2 min and treated with PBS containing 10 μg/ml RNase A (Nacalai Tesque). After the addition of 100 μg/ml propidium iodide (PI) (Wako Pure Chemical Industries, Tokyo, Japan), cell cycle parameters were assessed by a flow cytometric analysis using FACS Calibur as described previously [14 (link)].
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