HL-60 cells were cultured in 12-well plates at a density of 1 × 106 cells/well and incubated with 0.6 μM Ara-C or Ara-C@HFn with the equivalent amount of Ara-C for 48 h. The cell cycle was assessed using a DNA Content Quantitation Assay (Solarbio) through the flow cytometry system (Beckman, n = 3).
Dna content quantitation assay
Manufactured by Solarbio
Sourced in China
The DNA Content Quantitation Assay is a laboratory equipment used to measure the amount of DNA present in a sample. It provides a quantitative analysis of the DNA content, allowing researchers to determine the concentration of DNA in their experiments.
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Lab products found in correlation
Nuclear protein extraction kit, by Solarbio (2 mentions)
Flow cytometry system, by Beckman Coulter (1 mentions)
Annexin 5 apoptosis detection kit, by Beyotime (1 mentions)
Huvec cells, by American Type Culture Collection (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Solarbio (1 mentions)
Guava easycyte 6ht, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Solarbio (1 mentions)
Fr180204, by MedChemExpress (1 mentions)
Sp600125, by MedChemExpress (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Sb203580, by MedChemExpress (1 mentions)
5 fluorouracil 5 fu, by MedChemExpress (1 mentions)
Facscalibur, by BD (1 mentions)
10 protocols using dna content quantitation assay
1
Ara-C cytotoxicity in HL-60 cells
2
Cell Cycle Analysis by Flow Cytometry
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Cells were seeded in six-well plates and transfected with oligoribonucleotides. After 48 h, cells were harvested and cell cycle was analyzed by DNA Content Quantitation Assay (Cell Cycle) (Solarbio life sciences, Beijing, China) and Flow cytometry.
3
Cell Cycle Analysis by Flow Cytometry
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Cells were collected and fixed with 70% alcohol overnight at 4 °C. Cell cycle assays were performed by DNA Content Quantitation Assay (CA1510, Solarbio). Finally, cells were analyzed using BD FETASSA.
4
Phycocyanin Extraction and Cell Analysis
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The crude extract of R-phycocyanin was prepared on a laboratory scale from Porphyra haitanensis (the contents of crude protein 15.78 μg/mL). HUVEC cells were purchased from ATCC. Drosophila melanogaster was from Engineering Research Centre of Fujian-Taiwan Special Marine Food Processing and Nutrition.
Senescence-associated β-galactosidase (SA β-gal) staining, Hoechst and PI Staining Kit, Annexin V Apoptosis Detection Kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Cells counting kit-8 (CCK-8), the Mitochondrial Membrane Potential Assay Kit with JC-1, DNA content quantitation assay, and Nuclear Protein Extraction Kit were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). ECL was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
Senescence-associated β-galactosidase (SA β-gal) staining, Hoechst and PI Staining Kit, Annexin V Apoptosis Detection Kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Cells counting kit-8 (CCK-8), the Mitochondrial Membrane Potential Assay Kit with JC-1, DNA content quantitation assay, and Nuclear Protein Extraction Kit were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). ECL was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
5
PI Staining for Cell Cycle Analysis
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PI staining for flow cytometry was performed according to the user manual of DNA Content Quantitation Assay (Cell Cycle) from Solarbio (#CA1510). Briefly, OPCs from control or Tet1 cKO mice were harvested, washed in PBS, and fixed in cold 70% ethanol for 30 min at 4 °C. After washing twice in PBS, cells were treated with RNase and then stained with PI. With guava easyCyte6HT (Millipore), the forward scatter (FS) and side scatter (SS) were measured to identify single cells. For analysis, ModFit LT 5.0 software was used to make the PI histogram plot. Experiments were replicated three times.
6
Evaluating 18β-Glycyrrhetinic Acid's Cytotoxicity
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18β-Gly was obtained from Herbpurify Co., Ltd. (purity ≥ 98%; Chengdu, China), and it was dissolved with cell-level dimethyl sulfoxide (DMSO). The final concentration of DMSO is less than 0.5%, which can rule out the cytotoxic effect of the solvent on the cells. The Mitochondrial Membrane Potential Assay Kit (JC-1), DNA Content Quantitation Assay, and Nuclear Protein Extraction Kit were obtained from Solarbio (Beijing, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute-1640 (RPMI-1640), penicillin, and streptomycin were purchased from Gibco (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8), Apoptosis and Necrosis Assay Kit, Annexin V-FITC Apoptosis Detection Kit, and ROS Assay Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). All of the antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). 5-Fluorouracil (5-FU, purity ≥ 99.86%) and MAPK inhibitors (SB203580, inhibitor of p38; SP600125, inhibitor of c-Jun N-terminal kinase (JNK); FR180204, inhibitor of ERK) were obtained from MedChem Express (Princeton, NJ, USA). N-Acetyl-L-cysteine (NAC) and dimethyl sulfoxide were purchased from Sigma (St. Louis, MO, USA). Secondary antibodies were purchased from ZSGB-Bio, Inc. (Beijing, China).
7
Cell Cycle Analysis by Flow Cytometry
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The cellular cycle experiment was performed according to the protocol of the manufacturer’s DNA Content Quantitation Assay (Cell Cycle, CA1510, Solarbio). We washed the SPCA1 and A549 vector and SPCA1, A549 overexpressed cell lines with PBS once, collected it and centrifuged it at 1,500 rpm for 5 min, then used a cell counter to adjust the cell concentration to 1×106/mL, and took 1 mL of single-cell line suspension. After the single-cell suspension was centrifuged, we removed the supernatant, added 500 µL of 70% pre-cooled ethanol to the cells, and fixed it for 2 h to overnight, stored at 4 ℃, before washing off the fixative with phosphate buffered saline (PBS) before staining. If necessary, the cell suspension was filtered once with a cell sieve. A total of 100 µL of RNaseA solution was added to the cell precipitation, which resuspended the cells. Then, this solution was bathed in water at 37 ℃ for 30 min. We added 400 µL of propidium iodide (PI) staining solution and mixed well, incubated the solution at 4 ℃ for 30 min in the dark, tested the flow cytometer, and recorded the red fluorescence at the excitation wavelength of 488 nm. The experiment was carried out 3 times to ensure the reproducibility of the result.
8
Cell Cycle Analysis by Flow Cytometry
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Detection of cell cycle was performed with the DNA content quantitation assay (Solarbio) according to the instructions and then quantitatively analyzed by flow cytometry. 4×105 cells/well were pre-plated into 6-well plates, and then were treated with the indicated reagents for 24 h and 48 h, respectively. Adherent cells were collected, and washed twice with PBS. The cells were fixed with 75% pre-cooled ethanol at 4°C for 2 h, and were washed with PBS. Then, 100 μL of RNase A was used to treat cells to remove RNA interference (30 min, 37°C). Cells were incubated with PI in a dark room for 15 min, and then were detected with a flow cytometer.
9
Cell Cycle Analysis of PTX and PTX-TTHA Treatments
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Cell cycle assay was performed using the DNA Content Quantitation Assay (Solarbio, Beijing, China) [77 (link)]. MDA-MB-231 cells—after 48 h of PTX (0.1 nM) or PTX-TTHA (0, 0.1, 1 nM) treatment—were harvested and fixed with 70% ethanol at 4 °C overnight. Then, the fixed cells were incubated with RNase A solution at 37 °C for 30 min and stained with PI in the dark for 30 min at 4 °C. The red fluorescence was detected using a flow cytometer (BD FACSCalibur) at 488 nm and analyzed by FlowJo Software (Tree Star Inc, Ashland, OR, USA). The PTX-treated group was set as a positive control.
10
Cell Cycle Arrest and Nuclear Extraction in ISO-treated A549 Cells
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The cell cycle arrest of ISO-treated A549 cells was assessed by a DNA Content Quantitation assay (Solarbio Science &Technology Co., Ltd.). Briefly, the A549 cells were treated with 46.81 µM ISO for different periods of time (3, 6, 12 and 24 h ). After pre-cooling with 70% ethanol overnight, they were washed twice with phosphate-buffered saline (PBS), followed by the addition of 100 µl RNase A solution and the re-suspension of cells at 37˚C for 30 min. Finally, 400 µl PI staining solution were added, and the cells were incubated for 30 min at 4˚C. The data were then analyzed by flow cytometry.
Preparation of nuclear extracts. The Nuclear Protein Extraction kit was used to prepare the nuclear extract. The A549 cells were treated with 46.81 µM ISO for different periods of time (3, 6, 12 and 24 h), and then washed with PBS once; the cells were then centrifuged at 500 x g for 3 min in room temperature. After the cells were resuspended with 80 µl plasma protein extraction reagent, they were incubated on ice for 10 min. The cells were then centrifuged at 12,000 x g for 10 min at 4˚C and the supernatants were used as the cytosolic extract. The precipitate was then resuspended in 50 µl nuclear protein extraction reagent and incubated on ice for 10 min. The cells were then centrifuged at 12,000 x g for 10 min at 4˚C. The supernatant was used as the nuclear protein.
Preparation of nuclear extracts. The Nuclear Protein Extraction kit was used to prepare the nuclear extract. The A549 cells were treated with 46.81 µM ISO for different periods of time (3, 6, 12 and 24 h), and then washed with PBS once; the cells were then centrifuged at 500 x g for 3 min in room temperature. After the cells were resuspended with 80 µl plasma protein extraction reagent, they were incubated on ice for 10 min. The cells were then centrifuged at 12,000 x g for 10 min at 4˚C and the supernatants were used as the cytosolic extract. The precipitate was then resuspended in 50 µl nuclear protein extraction reagent and incubated on ice for 10 min. The cells were then centrifuged at 12,000 x g for 10 min at 4˚C. The supernatant was used as the nuclear protein.
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