Phenotyping of cell surface markers was performed by flow cytometry. The cells were stained with CD34-PE and CD45-FITC (all from BD Biosciences, USA), CD90-FITC (Dako, USA), CD73 PE (Abcam, USA) and isotype controls IgG1-FITC (Dako, USA), IgG1-PE (BD Biosciences, USA), and IgG2A-APC (BD Biosciences, USA). Flow cytometry data were acquired using a Guava EasyCyte 8HT flow cytometer and analysed using ExpressPro software (Merck Millipore, USA) comparing unlabelled, marker-labelled and isotype control populations in FL-1, FL-2 and FL-4 channels.
Expresspro software
Manufactured by Merck Group
Sourced in United States
ExpressPro Software is a laboratory data management tool designed to streamline the organization and analysis of experimental data. It provides a centralized platform for researchers to store, access, and share their findings effectively.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte flow cytometer, by Merck Group (6 mentions)
Guava easycyte 8ht, by Merck Group (1 mentions)
Cd45 fitc, by BD (1 mentions)
Cd34 pe, by BD (1 mentions)
Igg1 pe, by BD (1 mentions)
Cd73 pe, by Abcam (1 mentions)
Cd90 fitc, by Agilent Technologies (1 mentions)
Igg1 fitc, by Agilent Technologies (1 mentions)
Igg2a apc, by BD (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Cell cycle reagent, by Merck Group (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
11 protocols using expresspro software
1
Phenotyping Cell Surface Markers
2
Optimizing Quantum Dot Uptake by MSCs
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To estimate the optimal QD concentration for uptake experiments, MSCs were seeded at a density of 5 × 104 cells per well in a 12-well tissue culture polystyrene plate and labelled with QDs at various concentrations in the range of 0.5 to 64 nM for 6 h in complete or serum-free medium. To determine the accumulation dynamics, 8 nM or 16 nM QDs were applied to MSCs and incubated for 0.5, 1, 3, 6, 24 and 48 h in complete medium. The cells were subsequently harvested by trypsinization, centrifuged at 250g for 5 min and resuspended in 200 μL of PBS. The samples were acquired on a Guava EasyCyte 8HT flow cytometer and analysed using ExpressPro software (Merck Millipore, USA) in channel FL4, comparing unlabelled and labelled cell populations.
3
Flow Cytometric Detection of CD133 and pNF-κB
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Detection of CD133 and phosphorylated NF-κB was performed by flow cytometry as described elsewhere15 (link) with minor modifications. Briefly, 0.5 × 106 cells were plated in 2 ml of cell growth media in six-well plates and incubated overnight at 37°C and 5% CO2. Healthy, untreated A549 cells/A549-CS were used to detect CD133. For NF-κB detection, A549-CS were treated with IOX-101 for 24 h in a regular cell incubator. Cells were stained for 20 min in the dark with anti-CD133 FITC or phospho-NF-κB p65 PE antibodies in intracellular fixation and permeabilization buffer reagent (Thermo Scientific). After a couple of washes with washing buffer, the cells were resuspended in PBS and 5,000 events were acquired in a Guava easyCyte™ flow cytometer. Cells were gated for CD133 or phosphorylated NF-κB-positive events and analyzed using ExpressPro software from Millipore.
4
Measuring Intracellular ROS Levels
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The oxidant-sensitive dye dichloro-dihydro-fluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology) was used to detect intracellular ROS levels. Exponentially growing A2780 and A2780/PA cells were seeded in 6-well culture plates at a density of 3×105 cells/well. After 24 h of incubation, the fresh medium was changed and 400 ng/ml paclitaxel was added for 12 h. Then the cells were trypsinized and incubated in 2 ml DCFH-DA (10 µM) working solution for 20 min at 37°C in the dark, washed with PBS twice and then evaluated using a guava easyCyte flow cytometer (Millipore). Data analysis was performed using ExpressPro software (version 8.1; Millipore).
5
Cell Cycle Analysis of Renal Cancer Cells
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The assay was carried out with Guava® cell cycle reagent, according to the manufacturer’s instructions. Caki-1 and A-498 cells, at a density of 0.5 × 106 cells per well, were seeded in a 6-well plate and allowed to adhere for 24 h. FPMXY-14 at a concentration of 75 nM and 100 nM were added to Caki-1 and A-498 cells, respectively, followed by another incubation of 48 h. The media was aspirated, and cells were removed by trypsinization. After a couple of washes with sterile PBS, 50 µl cell cycle assay reagent was added, then incubated in the dark for 15 min washed with wash buffer two times and re-suspended in HBSS buffer. Ten thousand events were acquired on a Guava easyCyte™ flow cytometer, and the data were analyzed with ExpressPro Software from Millipore (Burlington, CA, USA). The percentage of cell populations at different cell cycle stages was presented.
6
Cell Cycle Analysis of A549 Cells
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A549 cells and A549-CS were incubated with DDP/GEM/IOX-101 at the desired concentrations for 48 h at 37°C and fixed with 70% ethanol until analysis. Further, cell cycle reagent from Millipore was used to stain the cells, and events were acquired on a Guava easyCyte™ flow cytometer. ExpressPro software from Millipore was used for the analysis.
7
Quantifying B-Raf and PI3K in Melanoma Cells
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A375 and G-361 cells were treated with 225 or 140 nM of CB-006-3, respectively, and incubated for 4 h in a 5% CO2 incubator at 37°C. After incubation, the cells were removed by trypsinization, washed twice with sterile PBS, and re-suspended in HBSS buffer. For B-RafV600E assay, both cells were treated with 0.50 μg/mL FITC-anti-B-RafV600E antibody, incubated for 30 min in the dark, washed twice with PBS, and resuspended in HBSS buffer. For PI3K assays, A375 and G-361 cells were treated with 0.7 μg/mL anti-PI3K CD or PI3K CG antibodies were added and incubated for 30 min in the dark. Ten thousand events were acquired using the Guava easyCyte flow cytometer, and the data were analysed with ExpressPro Software from Millipore (Burlington, MA, USA). The percentage of positive cells for each protein was presented.
8
Cell Cycle Analysis of Cancer Cells Treated with FCY-302
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Cancer cells were seeded at a density of 0.5 × 106 cells per well in a six-well plate and incubated with FCY-302 at desired concentrations for 72 h at 37°C in a 5% CO2 incubator. After incubation, cells were fixed in 70% ethanol and stored at −20°C until analysis. Cells were stained with Guava® Cell Cycle reagent according to the manufacturer’s instructions, and 10,000 events were acquired on a Guava easyCyte™ flow cytometer. Data were analyzed using Express Pro software (Millipore), and percent cell population in different cell cycle stages with respect to control was calculated.
9
Cell Cycle Assay of BRAF-Mutant Melanoma
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The assay was carried out using a cell cycle assay kit according to the manufacturer’s instructions. A375 and G-361 cells at a density of 0.5 × 106 cells/well were seeded in a 6-well plate and incubated for 24 h. After adding 100 nM/200 nM of CB-RAF600E-1, the cells were incubated for a period of 72 h. After washing twice with sterile phosphate-buffered saline (PBS), cells were treated with 50 μL cell cycle assay reagent, incubated in the dark for 15 min, washed twice with wash buffer, and resuspended in HBSS buffer. Ten thousand events were acquired on a Guava easyCyte flow cytometer, and the data were analysed with ExpressPro Software from Millipore (Burlington, MA, USA). The percentage of the cell population at the sub G0/G1 phase was presented.
10
Cell Cycle Analysis of A375 Cells
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The assay was performed using a cell cycle assay kit, according to the manufacturer’s instructions. A375-N or A375-R cells at a density of 0.5 × 106 cells/well were seeded in a 6-well plate and incubated for 24 h. After adding 100 nM or 200 nM CB-RAF600E-1, the cells were incubated for 72 h. After washing twice with sterile phosphate-buffered saline (PBS), cells were treated with 50 μL cell cycle assay reagent, incubated in the dark for 15 min, washed twice with wash buffer, and resuspended in HBSS buffer. Ten thousand events were acquired on a Guava easyCyte flow cytometer, and the data were analyzed with ExpressPro Software from Millipore. The percentage of the cell population in the sub G0/G1 phase is presented.
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