Imatinib-related apoptosis was assessed by seeding OSCC cells at a density of 1 × 104 cells per well in 6-well culture dishes and allowing them to adhere overnight. The cells were treated with different imatinib concentrations over 48h and were then harvested and washed with 1 × cold PBS solution. The washed cells were centrifuged, and the supernatant was discarded. Next, the cells were resuspended in 1 × annexin-binding buffer and stained with annexin V (Invitrogen, Thermo Fisher Scientific, USA) and propidium iodide (PI) at room temperature for 15 min to visualize apoptotic cells. The stained cells were analyzed using the FACS Verse Flow Cytometry system (BD Science, CA, USA).
Facsverse flow cytometry system
Manufactured by BD
Sourced in United States
The FACSVerse flow cytometry system is a compact and versatile instrument designed for multiparameter analysis of cells and other particles. It utilizes flow cytometry technology to rapidly detect and measure physical and fluorescent characteristics of individual cells or particles as they flow through a laser beam. The FACSVerse system is capable of detecting multiple fluorescent signals simultaneously, enabling detailed analysis of cellular properties and populations.
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Lab products found in correlation
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Fxcycle pi rnase staining solution, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Staining buffer, by BD (1 mentions)
Fixation buffer, by BD (1 mentions)
Aldefluor assay, by STEMCELL (1 mentions)
6 protocols using facsverse flow cytometry system
1
Imatinib-induced Apoptosis in OSCC
2
Cell Cycle Analysis of OSCC Cells
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For cell cycle analysis, OSCC cells were seeded at 1 × 105 cells per well in 6-well culture dishes and incubated overnight in a 37 °C incubator for cell attachment and growth. Then, the cells were treated with different imatinib concentrations over 24h and were then harvested and washed twice with cold 1× PBS. To fix the cells, they were treated with 70% ethanol and incubated overnight at -20°C. Next, the cells were washed with cold 1 × PBS to remove residual fixative and stained with 0.5 mL of the FxCycleTM PI/RNase Staining Solution (Thermo Fisher Scientific) to label DNA content and enable cell cycle analysis. The staining solution was added to each flow cytometry sample and incubated at room temperature for 15-30 min, protected from light. Then, the samples were analyzed using a FACS Verse Flow Cytometry System (BD Sciences, CA, USA).
3
Annexin V-FITC Apoptosis Assay
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The apoptosis of cells was detected by a FITC Annexin V Apoptosis Detection Kit (Beyotime, Hangzhou, China). Cells were digested with 0.25% trypsin. Annexin V-FITC and propidium iodide (PI) were added successively and incubated in the dark at room temperature for 5 min. Apoptotic cells were then detected using a FACSVerse flow cytometry system (BD Biosciences).
4
Transfection Efficiency Quantification
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Following a 42 h incubation period (Sections 2.5.3 and 2.5.4), cell populations were washed three times with PBS before addition of EpiLife imaging media (Life Technologies, UK) to permit live cell imaging. The presence of GFP and TdTomato in cell monolayers was visualised using the Leica DM IRB epifluorescence microscope and imaging system (Leica Microsystems Ltd., UK). The transfection efficiency of pEGFP-N1 and pIRES-TdTomato was determined by flow cytometry using the FACSVerse flow cytometry system (BD Biosciences, UK). To prepare for FACS analysis cells were washed with PBS and then treated with trypsin-EDTA at 37 °C for 10 min. The cell suspension was then re-suspended in growth medium, centrifuged (Thermo Fisher Scientific, UK) at 300 × g for 5 min and washed with PBS. Pelleted cells were fixed with 100 μL of fixation buffer (BD Biosciences, UK) for 15 min at 4 °C, washed and re-suspended in 200 μL of staining buffer (BSA), (BD Biosciences, UK) before flow cytometry analysis. GFP fluorescence was detected using a 527/32 filter, whilst TdTomato used a 568/42 filter (PE channel). On each sample, a minimum of 10,000 events was collected. Analysis of collected data was performed with the FlowJo Flow Cytometry Analysis Software for Mac Version 10. (Tree Star Inc., USA). The percentage of gated cells in each sample indicates the percentage of fluorescent cells.
5
ALDEFLUOR Assay for Stem Cell Identification
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The ALDEFLUOR assay was performed per the manufacturer’s instructions (StemCell Technologies, Vancouver, BC, Canada). Data acquisition was performed using a BD FACSVerse flow cytometry system (BD Biosciences).
6
Neutrophil Apoptosis Assay with Treatments
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Neutrophils were treated with 10 μM LTB4 alone or in combination with 30 μM calycosin and 8 μg/mL gallic acid in RPMI1640/10% FBS for 3, 6, 9, and 18 h. At the end of drug treatment, the cells were stained with Annexin V-FITC and PI according to manufacturer's instructions. Briefly, the cells were washed twice with ice-cold PBS and resuspended in 1x Binding Buffer at a concentration of 1 × 106 cells/mL. Cell suspensions (1 × 105 cells in 100 μL) were incubated with 5 μL of Annexin V-FITC and 10 μL PI in a 5 mL culture tube at room temperature for 20 min. The stained cells were immediately analyzed on BD FACSVerse flow cytometry system (San Jose, CA, USA).
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