Cells were separated with CD44 microbeads and resuspended in 500 μL PBS containing 5 μL Cell Cycle Assay Solution Blue (Dojindo Molecular Technologies) according to the manufacturer's instructions for 15 min at 37°C. A FACS Verse (BD Biosciences) was used to analyze stained cells filtered into a conical tube with a 35 μm nylon filter.
Cell cycle assay solution blue
Manufactured by Dojindo Laboratories
Sourced in Japan
The Cell Cycle Assay Solution Blue is a ready-to-use solution designed for the detection and analysis of cell cycle progression in various cell types. It provides a simple and reliable method to quantify the distribution of cells in different phases of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 fitc apoptosis detection kit, by Nacalai Tesque (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Facsverse, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 system, by BD (1 mentions)
Gallios ow cytometer, by Beckman Coulter (1 mentions)
8 protocols using cell cycle assay solution blue
1
Cell Cycle Analysis of CD44+ Cells
2
Cell Cycle Analysis of 3T3-L1 Cells
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After MDI induction, the 3T3-L1 cells were trypsinized for 5 min at 37 °C. Harvested cells were washed by PBS and fixed with 70% ethanol at 4 °C overnight. The cells were then wash by PBS three times and stained by Cell Cycle Assay Solution Blue (Dojindo) at 37 °C for 15 min. Cells were analyzed using LSRFortessa (BD Bioscience) equipped with 405 nm laser and 450/50 nm filter. Data were analyzed using FlowJo v10 software (BD Bioscience).
3
Cell Cycle and Apoptosis Analysis
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Cell Cycle Assay Solution Blue (C549, Dojindo) was used to measure the cell cycle according to the manufacturer’s instructions. Briefly, treated cells were synchronized at the G1 phase through serum starvation with 0.1% FBS-containing medium for 48 h. At 24 h after the release of serum starvation, the treated cells were collected, washed with PBS (−), and incubated with 5 μL cell cycle assay solution for 15 min at 37 °C. Thereafter, DNA content was determined based on staining intensity using a Gallios flow cytometer (Beckman Coulter, Miami, FL, USA). The annexin V-FITC Apoptosis Detection Kit (15342-54 Nacalai) was used to detect apoptosis by measuring annexin V and propidium iodide (PI)-positive cells following the manufacturer’s instructions. Briefly, cells were incubated for 96 h after siRNA transfection. To induce apoptosis, the cells were exposed to either 7.5 μM of gefitinib (078-06561, FUJIFILM) or 10 μM of cisplatin (P4394, Sigma-Aldrich) alone for 48 h after siRNA transfection. The treated cells were collected, washed with PBS (−), and incubated with 5 μL of annexin V-FITC solution and 5 μL of PI solution for 15 min. Thereafter, apoptotic cells were determined using a Gallios flow cytometer. Results were analyzed using the FlowJo software (Becton, Dickinson, Franklin Lakes, NJ, USA), after which the extent of apoptosis and cell cycle distribution were determined.
4
Cell Cycle Analysis via Flow Cytometry
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Cultured cells were transfected with the indicated siRNAs for 48 h using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After washing with PBS, unfixed cells were treated with Cell Cycle Assay Solution Blue (Dojindo, Kumamoto, Japan) at 37°C for 15 min. To stain the cells with propidium iodide (PI), they were fixed with 70% ethanol, and kept at −20°C before use. The cells were then incubated with 2 mg/ml RNase A at 37°C for 30 min and stained with PI at room temperature for 30 min. Subsequently, the cell suspensions were analyzed on the BD FACSCanto II system (Becton Dickinson, Franklin Lakes, NJ, USA). Data analysis was performed using FlowJo software (Becton Dickinson).
5
Cell Cycle Analysis Using Flow Cytometry
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Cell Cycle Assay Solution Blue (C549, Dojindo) was used according to the manufacturer’s instructions to analyze cell cycle. Briefly, treated cells were synchronized at the G1 phase by serum starvation with 0.1% FBS-containing medium for 48 h. After 24–36 h after release from serum starvation, the treated cells were collected, washed with phosphate-buffered saline (PBS), and incubated with 5 μL cell cycle assay solution for 15 min at 37°C. Thereafter, DNA content was determined by measuring staining intensity using a Gallios flow cytometer (Beckman Coulter, Miami, FL, USA). Results were analyzed using the FlowJo software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
6
Cell Cycle Analysis of Colon26 Cells
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Colon26 cells were seeded in 6-well culture plates at a density of 1 × 105 cells and incubated for 24 h. Fresh medium containing rbNPs or rb-sup (1,000 µg protein/mL) was added. After incubation for 6 h, the cells were washed with PBS and collected using a cell scraper. To analyze the cell cycle, cell nuclei were stained with Cell Cycle Assay Solution Blue (Dojindo Laboratories) according to the manufacturer’s protocol. The cells were analyzed using a BD FACSLyric flow cytometer, and cell cycle analysis was performed using FlowJo software ver8.7.
7
Cell cycle analysis after ADAM9 knockdown
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The effects of ADAM9 on the cell cycle in KK47 and T24 cells were measured using Cell Cycle Assay Solution Blue (DOJINDO, Kamimashiki, Japan). After ADAM9 knockdown, 1mL cell suspension having 1 × 105 cells/mL was prepared (n = 3) and centrifuged at 300 g for 5 min. The supernatant was removed, 1 mL PBS was added, and after resuspension, it was centrifuged at 300 g for 5 min. The supernatant was removed again; then, 0.5 mL PBS and 5 μL cell-cycle assay solution were added. After incubation for 15 min at 37 °C in shade, cells in each cell cycle were measured via flow cytometry.
8
Cell Cycle and Apoptosis Analysis
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Cell Cycle Assay Solution Blue (C549, Dojindo) was used to measure the cell cycle according to the manufacturer's instructions. Brie y, treated cells were synchronized at the G1 phase through serum starvation with 0.1% FBS-containing medium for 48 h. At 24 h after the release of serum starvation, the treated cells were collected, washed with PBS (-), and incubated with 5 µL cell cycle assay solution for 15 min at 37℃. Thereafter, DNA content was determined based on staining intensity using a Gallios ow cytometer (Beckman Coulter, Miami, FL, USA). The Annexin V-FITC Apoptosis Detection Kit (15342-54 Nacalai) was used to detect apoptosis by measuring annexin V and propidium iodide (PI)-positive cells following the manufacturer's instructions. Brie y, cells were incubated for 96 h after siRNA transfection. To induce apoptosis, the cells were exposed to either 7.5 µM of ge tinib (078-06561, FUJIFILM) or 10 µM of cisplatin (P4394, Sigma-Aldrich) alone for 48 h after siRNA transfection. The treated cells were collected, washed with PBS (-), and incubated with 5 µL of annexin V-FITC solution and 5 µL of PI solution for 15 min. Thereafter, apoptotic cells were determined using a Gallios ow cytometer. Results were analyzed using the FlowJo software (Becton, Dickinson, Franklin Lakes, NJ, USA), after which the extent of apoptosis and cell cycle distribution were determined.
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