Blood specimens for flow cytometric analysis of phosphorylated H2A.X expression in circulating peripheral blood mononuclear cells (PBMCs) were collected from the participants who agree to the measurement as mentioned above. Whole blood was mixed with the same volume of RPMI 1640 medium (HyClone, Logan, UT, USA) and gently laid on a histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). The sample was then centrifuged at 1800 rpm for 20 min at 10°C. After the separation, a thin layer of PBMCs was isolated and washed twice with RPMI 1640. The pellet was resuspended in RPMI 1640 with streptomycin [27 (link), 28 (link)]. Phosphorylation of histone H2A.X in PBMCs was measured with the Muse™ H2A.X activation dual detection kit (MCH200101, Millipore, Billerica, MA, USA). Briefly, isolated PBMCs were counted and diluted with assay buffer (5 × 106 cells/2.5 ml). After that, the cells were fixed, permeabilized, and incubated with antiphosphohistone H2A.X (Ser139, Alexa Fluor®555, part number CS208203, Millipore, Billerica, MA, USA) and anti-H2A.X (PECy5, part number CS209202, Millipore, Billerica, MA, USA), and then analyzed by the Muse cell analyzer (Millipore, Billerica, MA, USA) following the manufacturer's protocol. Each sample for the phosphorylated H2A.X measurement was prepared in triplicates; then, the average values were used in the statistical analysis.
Muse h2a x activation dual detection kit
Manufactured by Merck Group
Sourced in United States, Germany
The Muse H2A.X activation dual detection kit is a lab equipment product from Merck Group. It is designed to detect and quantify the activation of the H2A.X protein, which is a marker of DNA double-strand breaks. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
Muse cell analyzer, by Merck Group (2 mentions)
Alexa fluor 555, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Kinetoplast dna kdna, by TopoGEN (1 mentions)
Tetracycline tet, by Merck Group (1 mentions)
Etoposide vp 16, by Merck Group (1 mentions)
Vectashield mounting medium for fluorescence with dapi, by Vector Laboratories (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Karyomax colcemid solution, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Muse cell analyser, by Merck Group (1 mentions)
6 protocols using muse h2a x activation dual detection kit
1
Circulating PBMC H2A.X Phosphorylation Analysis
2
Azelastine-Induced DNA Damage Assessment
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To determine whether azelastine causes DNA damage, cells were fixed and permeabilized with Muse Fixation Buffer and Permeabilization Buffer reagents, followed by staining with anti-phospho-Histone H2A.X (Ser139) and anti-phospho-ATM (Ser1981) antibodies according to the instructions for the Muse H2A.X Activation Dual Detection kit (Millipore, Darmstadt, Germany).
3
Characterization of Topoisomerase II Isoforms
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Polyclonal antibodies for topo IIα and topo IIβ have been described previously (22 (link),23 (link)). The antibody to myc-tag protein (clone A46) was obtained from EMD Millipore (Billerica, MA). Anti-β-actin-peroxidase antibody (clone AC-15) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Geneticin and puromycin were obtained from Life Technologies (Carlsbad, CA, USA). Tetracycline (Tet) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The topo II catalytic inhibitor ICRF-193 was obtained from MP Biomedicals (Santa Ana, CA, USA). The topo II-targeting drugs etoposide (VP-16) and amsacrine (m-AMSA) were obtained from Sigma-Aldrich (St. Louis, MO) and the NCI, NIH, respectively. Stock solutions of these drugs were prepared in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) and stored at –20°C. Karyomax® colcemid solution was obtained from Life Technologies (Carlsbad, CA, USA). Muse H2A.X activation dual detection kit was obtained from EMD Millipore (Billerica, MA, USA). Vectashield mounting medium for fluorescence with DAPI was purchased from Vector Laboratories Inc. (Burlingame, CA, USA). Kinetoplast DNA (kDNA) was obtained from Topogen (Columbus, OH, USA).
4
DNA Oxidative Damage Induction in BCP-ALL and SEM Cells
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SEM cells were seeded onto 24‐well plate in RPMI medium supplemented with 10% FBS and penicillin/streptomycin solution at 0.2 × 106 cells·mL−1 density and subjected to AUR or ADE treatment for 24 h. BCP‐ALL primografts were seeded onto 48‐well plate at a density of 1 × 106 cells·mL−1 in SFEM II medium supplemented as described above. Induction of DNA oxidative damage was accessed by Muse® H2A.X Activation Dual Detection Kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer's protocol.
5
Quantifying DNA Damage in Frozen Cells
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Flow cytometry was used to quantify γH2AX phosphorylation in cells that were frozen with or without cryoprotectants. The Muse Cell Analyser (Merck Millipore) and Muse H2A.X Activation Dual Detection Kit (MCH200101, Merck Millipore), which discriminate between H2AX (−)/γH2AX (−) vs. H2AX (+)/γH2AX (−) vs. H2AX (−)/γH2AX (+) and H2AX (+)/γH2AX (+) cells, were used according to the manufacturer’s instructions. The cell state was analysed 1 h and 4 h after the cells were thawed.
6
Quantifying DNA Damage with Muse® H2AX Assay
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the Muse® Cell Analyzer (EMD Millipore) and Muse® H2AX Activation Dual Detection Kit (cat#MCH200101) were used to detect DNA damage. Live and dead cells were collected and combined, and cell samples were prepared and incubated with Muse® H2AX antibody cocktail for 30 minutes at room temperature (in the dark). Samples were acquired on Muse® Cell Analyzer and results were reported as percentages of activated H2AX (upper right quadrant).
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