The immunofluorescence method used in this study was described by Pandurangan et al. [23 (link)]. Paraffin-embedded colonic tissue sections with a thickness of 5 μm were deparaffinized in xylene and then rehydrated in a graded series of ethanol solutions. The slides were then blocked with 5% BSA in TBS for 90 min. The sections were then immunostained with rabbit anti-NF-κB (Santa Cruz Biotechnology, CA, USA) and anti-pSTAT3Y705 antibody (Cell Signaling Technology, CA, USA) diluted 1 : 100 with 5% BSA in TBS and incubated overnight at 4°C. After the sections were washed three times with TBS, the slides were incubated with goat and rabbit DyLight 550 secondary antibody (Thermo Scientific, Rockford, IL, USA) diluted 1 : 200 with TBS and incubated in the dark for 120 min at room temperature. The sections were then washed with TBS and incubated with the nucleus-specific counterstain propidium iodide (Nacalai Tesque, Japan) or 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) to stain the cell nuclei. The slides were mounted in an Ultracruz hard-set mounting medium (Santa Cruz Biotechnology Inc., Dallas, TX, USA), coverslipped, and visualized under a FSX100 fluorescent microscope (Olympus, Tokyo, Japan).
Propidium iodide
Manufactured by Nacalai Tesque
Sourced in Japan, United States
Propidium iodide is a fluorescent dye used for nucleic acid staining. It binds to DNA by intercalating between the bases. This dye is commonly used in flow cytometry and microscopy applications to detect and quantify cellular DNA content.
Automatically generated - may contain errors
Lab products found in correlation
Calcein am, by Nacalai Tesque (3 mentions)
Facscalibur, by BD (3 mentions)
Ultracruz hard set mounting medium, by Santa Cruz Biotechnology (2 mentions)
Dylight 550 secondary antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rnase a, by Nacalai Tesque (2 mentions)
Rnase a, by Merck Group (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Sh800 cell sorter, by Sony (2 mentions)
Rnase a, by Takara Bio (2 mentions)
Autophagy cytotoxicity dual staining kit, by Funakoshi (2 mentions)
Anti p stat3 y705 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti nf κb, by Santa Cruz Biotechnology (1 mentions)
Fsx100 fluorescent microscope, by Olympus (1 mentions)
Facsflow sheath fluid, by BD (1 mentions)
Facscalibur instrument, by BD (1 mentions)
Facsdiva software v8, by BD (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
34 protocols using propidium iodide
1
Immunofluorescence Staining for NF-κB and pSTAT3 in Colon Tissue
2
Cell Cycle Analysis by Flow Cytometry
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Flow cytometric analysis was performed as described previously (13 (link),14 (link)). HT29 and HCT116 cells were plated onto 10-cm dishes (5×105 cells per dish) in DMEM containing 5% FBS and grown at 37°C overnight to allow for cell attachment. Cells were then treated with DMSO (<0.5%) or 100 µg/ml crude acetone extract of MB at 37°C for 96 h, harvested, fixed with 70% ethanol, centrifuged (750 × g, at room temperature for 5 min), resuspended in 400 µl of PBS containing 2 mg/ml RNase (Nacalai Tesque Inc.), and stained with 400 µl of 0.1 mg/ml propidium iodide (Nacalai Tesque). The cell suspension was filtered through a 40 µm nylon filter (Ikemoto Scientific Technology Co. Ltd.). Samples of 20,000 cells suspended in BD FACSFlow™ Sheath Fluid at room temperature were then analyzed for DNA histograms (<1 min/run) and cell cycle phase distributions by flow cytometry using a FACSCalibur instrument (BD Biosciences), and the data were analyzed by a CELLQuest computer program (BD Biosciences), as previously described (13 (link)–15 (link)). Each assay was repeated in triplicate to confirm the results.
3
Quantifying Psychosine-Induced Multiploidy
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Multiploidy of the cells was examined as reported previously (Kanazawa et al., 2000 (link)). Briefly, cells treated with psychosine for 2 d were fixed in 70% ethanol overnight. After washing with PBS, the cells were incubated with RNase A (DNase-free; Nacalai Tesque, Kyoto, Japan) to remove RNA. The resultant cellular DNA was stained with propidium iodide (Nacalai Tesque) for subsequent flow cytometry (FCM) analyses using a FACScan or a FACSCalibur flow cytometer for FL-2 channel detection. To exclude doublets, cells were gated using the FL-2 Area and FL-2 Width. Average cell ploidy was calculated from the abundance ratio of each nuclear phase. In the multiploidization experiments, the comparison was made only using control cells cultured side by side. The PPIN value was developed to profile psychosine-induced multiploidy in six different B-cell lines, in which normalization of the cell-to-cell differences of psychosine sensitivity seemed appropriate. The concentration of psychosine to achieve maximum multiploidy was determined. Values of the maximum percentage of each cell with a ploidy >4N were divided by the concentration of psychosine to calculate the PPIN for normalization of cell variations in psychosine sensitivity and degree of multiploidy. When PPIN was not needed, average ploidy was calculated and is depicted on the upper right of the histograms.
4
Cell Cycle Analysis by Flow Cytometry
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The cells seeded in 75-cm2 flasks were exposed to 2 mM TCA or 300 µM deoxycholic acid (DCA, Sigma) for 24 h. They were harvested, washed with PBS, and fixed with 70% ethanol at room temperature for 30 min. The fixed cells were centrifuged and washed with PBS thrice. They were then resuspended in 0.5 mL of PBS containing 2 mg/mL RNase A (Sigma) at 37°C for 30 min and stained with 50 µg/mL propidium iodide (Nacalai Tesque) at 4°C for 1 h. The cellular DNA content was measured using FACSCalibur (Becton Dickinson, NJ, USA).
5
Cell Cycle Analysis of Maple Syrup Effects
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To analyze cell cycle distribution, DNA was stained with propidium iodide (PI; Nacalai Tesque, Inc., Kyoto, Japan). Briefly, DLD-1 cells were plated at a density of 2×105 cells/100-mm dish in culture medium. The next day, the culture medium was replaced with 1% (v/v) extra light grade maple syrup (Extra), dark grade maple syrup (Dark) or without syrup (control). Following 72 h of incubation at 37°C, the cells were washed with PBS and fixed in ice-cold 70% ethanol at 4°C for 2 h. The cells were subsequently treated with 0.25 mg/ml RNase in PBS for 60 min at 37°C, followed by staining with 50 µg/ml PI in PBS for 30 min at 4°C in the dark. Cell proportion in different phases of the cell cycle was determined using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using FACS DIVA software v8.0.1 (BD Biosciences).
6
BrdU Incorporation Assay
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Cells were pulsed with BrdU (10 μg/ml) (Sigma) for 30 min, stained with Alexa Fluor 488– or allophycocyanin-labeled antibodies to BrdU (BioLegend) and with propidium iodide (Nacalai), and then analyzed with a FACSVerse flow cytometer (BD).
7
Inhibition of mTOR Signaling Pathway
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Rapamycin and LY294002 were purchased from Calbiochem (San Diego, CA, USA); everolimus and AZD8055 from Selleckchem (Newmarket, UK) and MedChem Express (Monmouth Junction, NJ, USA). PP242 was synthesized by SAI Advantium Pharma Ltd (Hyderabad, Telangana, India). For in vitro assays, these chemicals were dissolved in 100% DMSO to a stock concentration of 10 mmol/L and stored at −20°C. FITC‐annexin V was purchased from BioLegend (San Diego, CA, USA). Propidium iodide (Nacalai Tesque) was dissolved in PBS to a stock concentration of 2 mg/mL. Rabbit anti‐phospho‐mTOR (Ser2448) (D9C2), rabbit anti‐phospho‐mTOR (Ser2481), rabbit anti‐mTOR (7C10), rabbit anti‐phospho‐Akt (Ser473) (D9E), mouse anti‐Akt, mouse anti‐phospho‐p70 S6 kinase (Thr389) (1A5), rabbit anti‐p70 S6 kinase, and mouse anti‐phospho‐p44/42 MAPK (Thr202/Tyr204) (E10) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐β‐actin (AC‐15, A5441) was purchased from Sigma‐Aldrich.
8
Retrograde Labeling of M1 Neurons
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Animals were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by an injection of glycerol (0.6 g/kg, intraperitoneally) and dexamethasone (1 mg/kg, intramuscularly), before being placed in a stereotaxic apparatus. Craniotomy was performed on M1 with a stainless steel trephine drill (Meisinger), and the dura mater was retracted. Filter papers with a size of 1 mm by 1 mm impregnated in fluorochrome Fast Blue (Dr Illing GmbH and Co. KG, Groß-Umstadt, Hesse, Germany; 2% in distilled water) were then placed on M1 for 5 min. Five days after the surgery, the mice were fixed as described above, and the brains were cut coronally into 50-μm-thick sections. The sections were counterstained with NeuroTrace 500/525 Green Fluorescent Nissl Stain (1:100 diluted; N21480, Thermo Fisher Scientific) and propidium iodide (10 μg/ml; 29037-76; Nacalai Tesque, Kyoto, Japan) in TBS containing 0.5% Triton X-100. The fluorescence signals were captured using a TCS SP8 confocal microscope (Leica Microsystems) with 10× objective lens (HC PL APO 10×/0.40 CS, NA = 0.40).
9
Palbociclib-Mediated Cell Cycle Arrest
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The cells were treated with 4 µM palbociclib (Sigma-Aldrich) 24 h prior to electroporation. The electroporated cells were further incubated in the recovery medium with or without 4 µM palbociclib for 3 days and then washed twice with SCM and cultured in SCM for 4 days, after which the cells were subjected to a proliferation assay. For the cell cycle analysis, cells were fixed with paraformaldehyde, treated with 200 µg/mL RNase A (Invitrogen), stained with 20 µg/mL propidium iodide (Nacalai Tesque), and analyzed with a FACS Aria III (BD) cell sorter using the FACS Diva software program (version 8.0.1; BD).
10
Cell Viability and Gene Expression Analysis
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Dichloromethane, methanol and dimethyl sulfoxide (DMSO) were purchased from Merck KGaA (Darmstadt, Germany). Acridine orange (AO) was purchased from Sigma (Sigma-Aldrich, St Louis, MO, USA). RPMI-1640, fetal bovine serum, trypsin, penicillin, propidium iodide (PI), RNase A and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were purchased from Nacalai Tesque (Kyoto, Japan). Real Genomics Total RNA extraction kit (RBC Biosciences, Taiwan) and GenomeLab GeXP Start Kit (Beckman Coulter, USA) were procured for this study. Tissue culture flasks and 96-well plates were acquired from TPP (Trasadingan, Switzerland).
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