The proportion of cells undergoing apoptosis during ANK-R mutant virus infection was determined using TUNEL staining to monitor nuclear DNA degradation and flow cytometric analysis. A549 cells (1×106) were seeded into 6 well plates. The following day, the cells were either mock infected or infected with vMyx-WT, vMyx-MT5KO, vMyx-148–150KO or vMyx-ANKsKO (MOI of 5). Both detached cells in the supernatant and trypsinized adherent cells were collected and pooled together. Fragmented DNA, a marker for apoptotic cell death, was stained using the In situ Direct DNA Fragmentation (TUNEL) Assay Kit (Abcam, ab66108) according to the manufacturer’s protocol. Anti-BrdU APC antibody (eBioscience) was used instead of anti-BrdU Red to allow the simultaneous detection of BrdU and DSred2. The cells were then characterized using a FACScalibur (Becton Dickinson) and analyzed by CELLQuest software. The collected data was analyzed using FCS Express 4 Flow Cytometry software (De Novo Software).
Anti brdu apc antibody
Manufactured by Thermo Fisher Scientific
The Anti-BrdU APC antibody is a fluorescently-labeled monoclonal antibody that specifically binds to bromodeoxyuridine (BrdU), a synthetic nucleoside that is incorporated into DNA during cell proliferation. This antibody can be used to detect and quantify cell division in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
In situ direct dna fragmentation tunel assay kit, by Abcam (1 mentions)
Fcs express 4 flow cytometry software, by De Novo Software (1 mentions)
Facscalibur, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rnase a, by Qiagen (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
2 protocols using anti brdu apc antibody
1
Apoptosis Quantification in Mutant Virus Infection
2
BrdU Incorporation and Cell Cycle Analysis
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Cells were incubated for 30 minutes with 10mM BrdU prior to harvest, and re-suspended in PBS, followed by dropwise addition of 9x volume of cold 70% ethanol and incubation on ice for 30 minutes. Cells were first re-suspended in denaturation buffer (PBS, 2M HCl, 0.5% Triton X-100) for 30 minutes at RT, followed by incubation in neutralization buffer (PBS, 0.1M Na 2 B 4 O 7 .10H 2 O, pH 8.5) for 30 minutes at RT, and then resuspended in PBS, 1% w/v BSA, 0.5% v/v Tween-20 containing anti-BrdU-APC antibody (eBioscience, 17-025-152), and incubated for 1 hour at RT. Cells were washed 3x in PBS and incubated in PBS with 5mg/ml RNase A (Qiagen) and 10mg/ml propidium iodide (Sigma Aldrich) for 30 minutes at RY prior to flow cytometry analysis using the CytoFLEX Flow Cytometer (Beckman Coulter).
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