To visualize dynamics of T cells and nuclei of ECs, a confluent monolayer of bEnd.3 cells treated with TNF-α and SDF-1α were stained with Hoechst 33342 (5 μg/ml, Life Technologies) for 20 min. Then, DO11.10 T cell blasts labeled with CellTraceTM CFSE (1 μM, Life Technologies) or Vybrant DyeCycleTM (0.5 μM, Life Technologies) were perfused on Hoechst-labeled bEnd.3 cells and each fluorescence image was sequentially acquired by time-lapse imaging for 15 min with 20 s interval.
Vybrant dyecycle
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Vybrant® DyeCycle™ is a fluorescent dye used in flow cytometry applications. It is designed to bind to DNA and emit fluorescence when excited, allowing for the analysis of cellular DNA content and cell cycle status.
Automatically generated - may contain errors
Lab products found in correlation
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc and pi apoptosis detection kit, by Beyotime (2 mentions)
Vybrant dyecycle, by BD (2 mentions)
Lsr fortessa facs analyzer, by BD (2 mentions)
Verapamil hydrochloride, by Merck Group (2 mentions)
Ly294002, by Merck Group (2 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
8 protocols using vybrant dyecycle
1
Visualizing T-cell and EC Dynamics
2
Neutrophil G-CSFR Modulation by R848
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Phenotypic studies were performed exactly as previously described8 (link). For G-CSFR staining, 105 neutrophils, incubated with or without 5 μM R848 for the indicated times, were harvested and stained for 15 min at room T with 1:20 PE anti-human CD114 (G-CSF-R) mAb (clone LMM741, Biolegend, San Diego, CA, USA), or with 1:20 PE control mouse IgG1 (BD Biosciences, San Jose, CA, USA). Then, cells were washed and stained for Vybrant DyeCycleTM (Life Technologies, Carlsbad, CA, USA) to discriminate cells that were alive, as described below. Sample fluorescence was measured by a seven-color MACSQuant Analyzer (Miltenyi Biotec), while data analysis was performed by using FlowJo software Version 8.8.6 (Tree Star, Ashland, OR, USA).
3
Cell Cycle and Apoptosis Analysis
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SNU-387 and Huh7 cells with transfection were seeded into 6-well plates (2 × 105 cells/well). After culturing for 24 h, cell cycle was detected using cell cycle analysis kit (Vybrant DyeCycle; Invitrogen) in line with the manufacturer’s guideline and analyzed by flow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
SNU-387 and Huh7 cells with transfection for 48 h were collected and washed with phosphatic buffer saline (PBS). Then, cells (6 × 104) were used to conduced apoptosis analysis using the Annexin V-FITC and PI Apoptosis Detection Kit (FITC: fluorescein isothiocyanate; PI: propidium iodide) (Beyotime) following the protocol. The apoptotic cells were analyzed by flow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
SNU-387 and Huh7 cells with transfection for 48 h were collected and washed with phosphatic buffer saline (PBS). Then, cells (6 × 104) were used to conduced apoptosis analysis using the Annexin V-FITC and PI Apoptosis Detection Kit (FITC: fluorescein isothiocyanate; PI: propidium iodide) (Beyotime) following the protocol. The apoptotic cells were analyzed by flow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
4
Cell Cycle Analysis of Anticancer Compounds
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Extracts from Passiflora edulis fruit (PEF), Annona muricata leaves (AML), Annona muricata seeds (AMS) that displayed the best cytotoxicity as well as doxorubicin were used to treat CCRF–CEM cells (1 × 106) at their IC50 values. Thus, CCRF–CEM cells were cultured in RPMI medium as described above, in the presence of each sample at a concentration corresponding to the IC50 values obtained in the cell line. The cell cycle was then analyzed after incubation for 24, 48 and 72 h. All reagents, experimental conditions and apparatus were identical to those previously reported (Kuete et al. 2013a (link); Dzoyem et al. 2013 (link)). Briefly, cell cycle analysis was performed by flow cytometry using Vybrant® DyeCycle™ (Invitrogen, Darmstadt, Germany). Cells were measured after Vybrant® DyeCycle™ Violet staining (30 min at 37 °C) on a LSR-Fortessa FACS analyzer (Becton–Dickinson, Heidelberg, Germany) using the violet laser. Vybrant® DyeCycle™ Violet stain was measured with 440 nm excitation. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate.
5
HTLV-I-transformed Cell Culture Protocol
6
Cell Cycle Analysis of Cytotoxic Extracts
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Extracts from Albizia adianthifolia roots (AAR) and Alchornea cordifolia leaves (ACL) that displayed the best cytotoxicity as well as doxorubicin were used to treat CCRF-CEM cells (1 × 106) at their IC50 values. The cell cycle was then analyzed after incubation for 24 h, 48 h and 72 h. All reagents, experimental conditions and apparatus were identical to those previously reported [12 (link), 16 (link)]. Briefly, cell cycle analysis was performed by flow cytometry using Vybrant® DyeCycle™ (Invitrogen, Darmstadt, Germany). Cells were measured after Vybrant® DyeCycle™ Violet staining (30 min at 37 °C) on a LSR-Fortessa FACS analyzer (Becton-Dickinson, Heidelberg, Germany) using the violet laser. Vybrant® DyeCycle™ Violet stain was measured with 440 nm excitation. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate.
7
HTLV-I-transformed Cell Culture Protocol
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HTLV-I-transformed cell lines ED, MT1, ATL-T, and ATL-25 were cultured in RPMI-1640 with 10% fetal bovine serum, L-glutamine, 100 U/ml penicillin and streptomycin and maintained in 5% CO2 at 37 °C. Vybrant® DyeCycle™ (DCV) was obtained from Invitrogen. Verapamil hydrochloride was purchased from Sigma-Aldrich. ED or MT1 cells were treated with either 10 µM LY294002 (Sigma-Aldrich, St Louis, MO) for 3 days or 1µM gamma-secretase inhibitor (GSI, Calbiochem) for 5 days as indicated in the figure legends.
8
Cell Cycle and Apoptosis Analysis
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SNU-387 and Huh7 cells with transfection were seeded into 6-well plates (2 × 10 5 cells/well). After culturing for 24 h, cell cycle was detected using cell cycle analysis kit (Vybrant DyeCycle; Invitrogen) in line with the manufacturer's guideline and analyzed by ow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
SNU-387 and Huh7 cells with transfection for 48 h were collected and washed with phosphatic buffer saline (PBS). Then, cells (6 × 10 4 ) were used to conduced apoptosis analysis using the Annexin V-FITC and PI Apoptosis Detection Kit (FITC: uorescein isothiocyanate; PI: propidium iodide) (Beyotime)
following the protocol. The apoptotic cells were analyzed by ow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
SNU-387 and Huh7 cells with transfection for 48 h were collected and washed with phosphatic buffer saline (PBS). Then, cells (6 × 10 4 ) were used to conduced apoptosis analysis using the Annexin V-FITC and PI Apoptosis Detection Kit (FITC: uorescein isothiocyanate; PI: propidium iodide) (Beyotime)
following the protocol. The apoptotic cells were analyzed by ow cytometry using Attune™ NxT Acoustic Focusing Cytometer (Invitrogen).
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