Cell cycle distribution was analyzed by measuring the DNA content using flow cytometry. Exponentially growing transduced cells were pelleted by centrifugation, washed with PBS (Sigma-Aldrich), resuspended in ice-cold 70% ethanol (Sigma-Aldrich) in PBS for fixation, and were maintained at 4 °C for 24 h. Prior to analysis, fixed cells were washed with phosphate-citrate buffer (Sigma-Aldrich) and incubated with propidium iodide staining solution (Sigma-Aldrich) containing 100 μg/mL of RNase (Qiagen) for 30 min. Cellular DNA content was measured using a BD FACSCantoTM platform (BD Biosciences) and analyzed by using FlowJo® software (version 10.0.6, FlowJo).
Propidium iodide staining solution
Manufactured by Merck Group
Sourced in United States
Propidium iodide staining solution is a fluorescent dye used to stain nucleic acids. It intercalates with DNA and emits red fluorescence when excited by appropriate wavelengths of light. The solution is commonly used in flow cytometry and other applications to identify and quantify cells at different stages of the cell cycle.
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Lab products found in correlation
Rnase a, by Merck Group (3 mentions)
Collagenase, by Merck Group (2 mentions)
Cd31 pe cy7, by Thermo Fisher Scientific (2 mentions)
F4 80 apc cy7, by BioLegend (2 mentions)
F4 80 apc, by Thermo Fisher Scientific (2 mentions)
Sca 1 fitc, by Thermo Fisher Scientific (2 mentions)
Sytox blue, by Thermo Fisher Scientific (2 mentions)
Cd45 pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd206 alex647, by Bio-Rad (2 mentions)
Sytox blue, by Tree Star (2 mentions)
Facsaria cell sorter, by BD (2 mentions)
Cd11c pe, by BD (2 mentions)
Facsuite software, by BD (2 mentions)
Cellquest pro software, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Rnase, by Qiagen (1 mentions)
Ethanol, by Merck Group (1 mentions)
Phosphate citrate buffer, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
15 protocols using propidium iodide staining solution
1
Cell Cycle Analysis by Flow Cytometry
2
Annexin V and PI Apoptosis Assay
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To assess the apoptosis of cancer cells, annexin V and propidium iodide (PI) staining assay was performed. After 24 h of treatment or control, adhesive cells were harvested through detachment by 0.25% trypsin and centrifugation at 1400 rcf for 10 min. After resuspending cells with 1 × Binding Buffer, 5 µl of fluorochrome-conjugated annexin V (Sigma-Aldrich, Saint Louis, USA) was added into 100 µl of cell suspension to stain intracellular phosphatidylserine (PS). Incubation was performed in dark under room temperature. 5 µl of propidium iodide staining solution (Sigma-Aldrich, Saint Louis, USA) was added into 100 µl of cell suspension. Flow cytometry (FACS Calibur, Becton Dickinson, Sunnyvale, CA, USA) was used to detect the percentage of apoptotic cells by FlowJo software (Treestar, Ashland, USA).
3
Flow Cytometric Analysis of Adipose Tissue
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Stromal vascular fraction (SVF) was obtained from pgWAT by treatment with 2 mg/mL collagenase (Sigma) for 45 min at 37 °C. The isolated SVF was resuspended in cold Hank’s balanced salt solution (HBSS) with 2% fetal bovine serum (FBS). Cells were incubated with CD45-PE-Cy7 (eBiosciences), F4/80-APC-Cy7 (BioLegend), CD206-Alex647 (Serotec, Inc.) and CD11c-PE (BD Pharmingen) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Sytox Blue (Thermo Scientific). Cells were analyzed on a BD FACSAria cell sorter after selection by forward scatter and side scatter, followed by exclusion of dead cells with Sytox Blue staining, and analyzed for cell-surface markers using FlowJo software (Tree Star). M1 or M2 macrophages were identified as F4/80-positive/CD11c-positive/CD206-negative or F4/80-positive/CD11c-negative/CD206-positive cells, respectively. The data are shown as the percentage of M1 and M2 macrophages. For sorting preadipocytes, endothelial cells and macrophages, cells were incubated with CD31-PE-Cy7 (eBiosciences), F4/80-APC (eBiosciences), and Sca-1 FITC (eBiosciences) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Propidium Iodide Staining Solution (Sigma). Gating strategies are presented in Supplementary Figure 12 and Supplementary Figure 13 .
4
Flow Cytometric Analysis of Adipose Tissue
5
Single-cell Dissociation and FACS Analysis
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Before FACS analysis, cells were incubated in Accutase (Nacalai Tesque) at 37°C for an hour for single-cell dissociation. Following dissociation, the cells were suspended in FACS buffer consisting of 1% inactivated fetal bovine serum (FBS), 5 mM EDTA (Sigma) and 10 μM Y-27632 in 1× PBS buffer, supplemented with 0.1% Propidium iodide staining solution (Sigma) for removing dead cells. FACS analysis was performed using the FACSAria III cell sorter (BD) with the FACSuite software (BD) following the manufacturer’s instructions.
6
Cell Synchronization and DNA Content Analysis
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Cells (1×105) were cultured in each well of 6-well plates till 60-70% confluence with normal culture medium. Cells were synchronized by the replacing the medium containing 0.1% FBS for 24 hours, and then replacing medium containing 1% FBS with or without EGF/TPA and ISO, and cultured for another 24 hours as indicated in figure legends. The cells were harvested and fixed with 3 ml of ice-cold 80% ethanol overnight. The fixed cells were washed twice with PBS, and then suspended in Propidium Iodide staining solution (Propidium Iodide 50 mg /ml, RNAse A 10 mg /ml, and 0.1% Triton X-100) (Sigma-Aldrich Chemical, St. Louis, MO) for at least 1 hour at 4°C. The DNA content was determined by means of Flow Cytometric Analysis with the Epics XL FACS (Beckman Coulter, Miami, FL) and EXPO 32 software as described previously [45 (link), 51 , 55 (link)].
7
Quantifying NK Cell-Induced Apoptosis
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To assess cell death in target cells, mEmerald-Lifeact+ HG-3, PGA-1, JVM-3 or MEC-1 target cells were incubated for 45 min with CD56-PE/Cy7-labeled NK-92MI cells. Cells were washed with cold Annexin V binding buffer (Biolegend, cat. # 422201) twice. Afterwards, cells were resuspended in 100 μl Annexin V binding buffer with 5 μl Alexa Fluor® 647 Annexin V (Biolegend, cat. #640912) and 5 μl propidium iodide staining solution (Sigma-Aldrich, cat. #P4864) per million cells. Cells were incubated for 15 min at RT in the dark, before addition of 400 μl Annexin V binding buffer and analysis by flow cytometry on a CytoFLEX (Beckman Coulter). Generated data were analyzed with FlowJo v10.6.2. software.
8
Flow Cytometric Analysis of Apoptosis
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flow cytometry was used to analyze the effects of VPA and MT on cell apoptosis using an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (eBioscience, San Diego, USA)
[28] (link). After treatment with VPA and MT, HT-22 cells were collected and then centrifuged at 400
g for 5 min. Cells were arranged into three groups as follows: (1) control group, (2) VPA group, and (3) VPA+MT group. Cells were subsequently incubated with 5 μL of annexin V-FITC and 10 μL of propidium iodide staining solution (Sigma-Aldrich) for 15 min at room temperature in the dark, and cell apoptosis in each group was detected by flow cytometry (BD Biosciences, San Jose, USA).
[28] (link). After treatment with VPA and MT, HT-22 cells were collected and then centrifuged at 400
g for 5 min. Cells were arranged into three groups as follows: (1) control group, (2) VPA group, and (3) VPA+MT group. Cells were subsequently incubated with 5 μL of annexin V-FITC and 10 μL of propidium iodide staining solution (Sigma-Aldrich) for 15 min at room temperature in the dark, and cell apoptosis in each group was detected by flow cytometry (BD Biosciences, San Jose, USA).
9
Flow Cytometric Analysis of cjESCs
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FACS analysis was performed using FACSVerse flow cytometer (BD) with FACSuite software (BD) according to the manufacturer’s instructions. In brief, the cjESCs were expanded following transient selection, and the cells were suspended in FACS buffer consisting of fetal bovine serum (1%), EDTA (5 mM) and Y-27632 (10 μM) in 1x PBS buffer. 0.1% Propidium iodide staining solution (PI; Sigma) was used to remove dead cells.
10
Cell Proliferation and Cell Cycle Analysis
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Cells were seeded at equal numbers 96-well plates and exposed to hormones as indicated and proliferation was measured using 5-bromo-2′-deoxyuridine (BrdU) proliferation kit according to the manufacturer's guidelines (BrdU cell proliferation ELISA, Roche, Mannheim, Germany).
For cell cycle analyses, cells were seeded at 100,000 cells/100 mm dishes and treated for 7 days, fixed in ethanol for 1 hour at 4°C, centrifuged and re-suspended in 1ml of propidium iodide staining solution (40μg/ml) (Sigma-Aldrich). 50μl of RNase A (10μg/ml) (Sigma-Aldrich) was added and the cells were incubated for 4 hours. Data was acquired on FACS Gallios (Beckman Coulter) and analyzed with ModFit LT (version 4, Verity Software).
For cell cycle analyses, cells were seeded at 100,000 cells/100 mm dishes and treated for 7 days, fixed in ethanol for 1 hour at 4°C, centrifuged and re-suspended in 1ml of propidium iodide staining solution (40μg/ml) (Sigma-Aldrich). 50μl of RNase A (10μg/ml) (Sigma-Aldrich) was added and the cells were incubated for 4 hours. Data was acquired on FACS Gallios (Beckman Coulter) and analyzed with ModFit LT (version 4, Verity Software).
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