The induction of apoptosis in the species studied was based on using CellEvent (ThermoFisher, São Paulo, SP, Brazil), following the manufacturer’s restrictions for detecting caspase action. Samples were prepared following the pattern described in the previous tests, and 9 μL of CellEvent® reagent (ThermoFisher, São Paulo, SP, Brazil) was added. Finally, washes and centrifugations were performed as before. Finally, the treatments were observed with a microscope (Olympus System BX60) using an excitation wavelength of 342 nm and an emission wavelength of 441 nm.
Cellevent
Manufactured by Thermo Fisher Scientific
Sourced in Brazil, United States
CellEvent is a fluorescence-based reagent that detects DNA fragmentation, a hallmark of apoptosis. It comprises a cell-permeable dye that binds to DNA upon cleavage, allowing for the identification and quantification of apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
System bx60, by Olympus (2 mentions)
Cytation 3 multi mode microplate reader, by Agilent Technologies (1 mentions)
Facscan, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Draq5, by Biostatus (1 mentions)
Biorad s3 sorter, by Bio-Rad (1 mentions)
40 μm nylon mesh, by BD (1 mentions)
Allstars hs cell death sirna, by Qiagen (1 mentions)
Uorescence microscope, by Nikon (1 mentions)
Round bottom ultra low attachment plate, by Corning (1 mentions)
15 protocols using cellevent
1
Detecting Apoptosis via CellEvent Reagent
2
Panx1 Knockout Adipose Tissue Analysis
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White adipose tissue (WAT) was isolated from WT and Panx1 KO mice after a 15-week high fat diet, by incising the abdomen of the mouse and excising all visible fat. Adipose tissue was fixed in 10% neutral buffered formalin overnight, processed at the Robarts Research Pathology facility and embedded in paraffin. Paraffin sections were taken at 6 µm thickness, sections were deparaffinized and immuno-labeled with Panx1-CT primary antibody (stock 1 μg/μl) in a 1:500 dilution as described previously by Penuela et al.37 (link). For proliferation studies, cells were labelled with active cell cycle phase specific marker Ki-67 in a 1:1000 dilution (Abcam, Cambridge, UK), and for cell death assays cells were labelled with CellEvent (Thermo Fisher Scientific) Caspase-3 Green Detection Reagent following manufacturer’s protocols, and counterstained with Hoechst nuclei stain in a 1:1000 dilution, as described previously26 (link).
3
Apoptosis Quantification via Cell Death Markers
4
Caspase Activity Measurement in Cryptococcus
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The caspase activity was measured after cell incubation for 24 h, in the presence and absence of synthetic peptides and ITR, according to the methodology described by Qorri and Harless [13 (link)]. After incubation for 24 h, as described above, the samples were washed three times with sterile 0.15 M NaCl and centrifuged (5000× g 5 min at 4 °C) to remove the YPD medium. The samples were washed and centrifuged as described above, and the cells were incubated with 3 μL of CellEvent® (ThermoFisher, São Paulo, SP, Brasil) for 30 min in the dark at room temperature (22 °C ± 2). Then, the samples were washed and centrifuged again. Finally, the cryptococcal cells were observed under a fluorescence microscope (Olympus System BX60), with an excitation wavelength of 342 nm and emission wavelength of 441 nm.
5
Evaluating Apoptosis via Caspase 3/7
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The activation of caspase 3/7 was assessed to evaluate if the cytotoxic core of MCTs carry features of apoptosis. The CellEvent™ caspase-3/7 green reagent is intrinsically non-fluorescent, as the DEVD peptide inhibits binding of the dye to DNA. However, upon activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved, and the free dye can bind the DNA, generating a bright green fluorescence. The CellEvent™ (Thermo Fisher Scientific R37111) ready to use reagent (25 µl) was added to each well containing spheres on day 4. After 30 min of incubation at 37 °C, images were taken using the Bio-Tek Cytation3 Multi-mode microplate reader (Agilent).
6
Apoptosis detection protocol
7
Apoptosis Quantification via Cell Death Markers
8
Apoptosis Induction After UV Irradiation
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To follow the effect of treatment on the induction of apoptosis post UV irradiation, CellEvent (Thermofisher Scientific) was utilized, which allows the measurement of caspase 3/7 activity. For that, cells were seeded in 100 mm dishes, treated with the molecules, irradiated, then Cell Event was added post UV and incubated for 24 h. The cells were then collected and PI was added before their analysis by flow cytometer (FACScan, BD LSRII flow cytometer, BD Biosciences). The post analysis was carried using flowing software [39 ] (Turku Bioimaging, Finland).
9
Apoptosis Quantification via Cell Death Markers
10
Quantifying Apoptosis in ZIKV-Infected Cells
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Staining for caspase-3/7 (CellEvent, Thermo Fisher) and propidium iodide (PI) was performed in 1 Â 10 5 tumor cells, 72 hours after infection with ZIKV BR , following the manufacturer's protocol. Flow cytometry analysis was performed using FACS Aria II (BD) collecting 10,000 events per run. Analysis was carried using FlowJo software.
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