Intracellular ROS levels were examined using chloromethyl-2, 7-dichlorofluorescin diacetate (DCFH-DA; Sigma-Aldrich) via MACSQuant Analyzer flow cytometry (North Rhine-Westphalia, Germany). Briefly, CHO-K1 cells were cultured with or without 50 μg/ml BRD125-EPS. After 24 hours, the cells were washed with phosphate-buffered saline (PBS), followed by incubation with 20 μM DCHF-DA at 37°C in the dark for 1 h. The cells were harvested and immediately exposed to 4 Gy of X-ray (160 kV, 1 mA). After irradiation, the cells were analyzed by flow cytometry. At least 10,000 events were analyzed.
Macsquant analyzer
Manufactured by Merck Group
Sourced in Germany
The MACSQuant® Analyzer is a flow cytometry instrument designed for high-performance cell analysis and sorting. It provides accurate and reliable data acquisition, processing, and analysis capabilities.
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Lab products found in correlation
3 protocols using macsquant analyzer
1
Intracellular ROS Levels Examination
2
Cytotoxic Assay for CAR T-Cell Elimination
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The elimination of CAR T cells by αE-tag CAR T cells was assessed using a previously established flow cytometry-based assay [33 (link), 34 (link)]. To distinguish effector from target cells, the latter were labeled with 10 µM cell proliferation dye eFluor™450 (eBioscience, ThermoFisher Scientific) according to the manufacturer’s instructions. The next day, effector cells (αE-tag CAR T cells) were incubated with eFluor™450-stained CAR 28/ζ, ΔCAR 28/ζ or CAR Stop T cells at indicated effector to target cell (E:T) ratios. After indicated time points, cocultures were carefully resuspended and an aliquot of 25 µl diluted 1:4 with 1 µg/ml propidium iodide (Sigma-Aldrich, Munich, Germany) was measured at a MACSQuant® Analyzer. After exclusion of doublets and dead cells, cells/ml were assessed for both effector and target T cells separately. Finally, cells of one triplet were pooled, stained for intracellular expression of GzmA and GzmB, and analyzed by flow cytometry.
The antitumor activity of αE-tag CAR T cells was determined in standard chromium-51 release assays as previously reported [25 (link)].
The antitumor activity of αE-tag CAR T cells was determined in standard chromium-51 release assays as previously reported [25 (link)].
3
Cell growth and cell cycle analysis
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For growth curve experiments, 70000 Rat HO15.19 cells were plated in triplicate in 6-well plates and counted every 3 days for 9 days. Similarly, 70000 3T9 cells expressing the various forms of MycER were plated in the presence or absence of 400 nM OHT, and counted every 2 days up to day 6. In the experiments performed with the cb9 Δmyc cells, 80000 cells per well were plated in presence of doxycycline for 2 days, then counted and re-plated with or without doxycycline, every two days for the following 10 days. For colony forming assays (CFA), for all cell lines, 10000 cells were plated in 10 cm dishes, let grow for 6-11 days and stained with crystal violet.
For cell cycle analysis, cells were incubated with 33 µM BrdU for 20 min, harvested and washed in PBS, and fixed in ice-cold ethanol. Upon DNA denaturation with 2N HCl for 25 minutes, cells were stained with an anti-BrdU primary antibody (BD Biosciences, #347580) and an FITC-conjugated anti-mouse secondary antibody (Jackson Immunoresearch, # 715-545-150).
DNA was stained by resuspending the cells in 2.5 µg/ml Propidium Iodide (Sigma) overnight at 4°C before acquisition with a MACSQuant® Analyzer.
For cell cycle analysis, cells were incubated with 33 µM BrdU for 20 min, harvested and washed in PBS, and fixed in ice-cold ethanol. Upon DNA denaturation with 2N HCl for 25 minutes, cells were stained with an anti-BrdU primary antibody (BD Biosciences, #347580) and an FITC-conjugated anti-mouse secondary antibody (Jackson Immunoresearch, # 715-545-150).
DNA was stained by resuspending the cells in 2.5 µg/ml Propidium Iodide (Sigma) overnight at 4°C before acquisition with a MACSQuant® Analyzer.
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