Foreword: Tumor necrosis factor (TNFα) is a key cellular inflammatory factor that regulates a variety of cellular events through many cellular pathways. This factor is well known for causing apoptosis in hematopoietic cells, such as U937, by activating the cascade of intracellular caspase. On the other hand, granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the members of hematopoietic growth factor and promotes proliferation and/or differentiation. However, it has been reported that U937 cells respond to GM-CSF and undergo growth inhibition and apoptosis. Because both TNFα and GM-CSF cause apoptosis in U937 cells, there is a different time course between the two cytokines. When the two are combined, a high degree of synergy between the two cytokines will be observed. The EarlyToxTM Cell Viability Kit is one of a class of reagents for evaluating cell growth conditions, including cell viability and various apoptotic events. These reagents were designed as homogeneous wash-free experiments and optimized for fluorescent microplate readers. Here, we used three different kits, Live/Dead assay, Caspase-3/7 R110 assay and Caspase-3/7 NucView 488 assay to detect the effect of TNFα and GM-CSF on U937 cells (Figure 1). • The EarlyToxTM Live/Dead Test Kit contains two markers for live or dead cells in mammals. Calcein AM is a marker widely used in living cells, and ethidium bromide dimer-III (EthD-III) enters dead cells through damaged cell membranes to bind to DNA to produce bright red fluorescence. • The EarlyToxTM Caspase-3/7 R110 Assay Kit provides a one-step, homogeneous assay that requires only cell lysis to perform endpoint analysis of the apoptotic process. • The EarlyToxTM Caspase-3/7 NucView 488 Assay Kit detects apoptotic cells in an intact population by using NucView 488 Caspase-3 substrate. This reagent is non-toxic to cells and can be used for dynamic studies of apoptosis. In this cell-based experiment, the fluorescence signal is detected by the upper reading domain scanning function of the SpectraMax® i3x microplate reader, and the average signal at 12 positions at the bottom of the well is obtained to avoid potential cell growth unevenness. The difference between the hole and the hole. material: • U937 cells (ATCC Cat.# CRL-1593.2) • Cytokines: • Human TNFα (Peprotech Cat# 300-01) • Human GM-CSF (Peprotech Cat# 300-03) • EarlyTox Cell Viability Kit (Molecular Devices, LLC): • EarlyTox Live/Dead Test Kit (Cat.# R8340) • EarlyTox Caspase-3/7 R110 Test Kit (Cat.# R8346) • EarlyTox Caspase-3/7 NucView 488 Test Kit (Cat.# R8348) • PBS•96-well, flat bottom, bottom through, black TC-treated microplate (Corning cat. #3904) • SpectraMax i3x Multi-Mode Microplate Reader and SpectraMax® MiniMax 300 Imaging Cell Counter method: Cell culture and processing U937 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and penicillin/streptomycin. On the day of the experiment, cells were seeded at 100,000 cells/80 μL/well. 20 μL of 5X concentration cytokine was added to each well to a final concentration of 1X and a volume of 100 μL. Incubate for 24 or 48 hours at 37C, 5% CO2 Cell viability and apoptosis assay After the treatment, the prepared reagent was added to each well as described below. Live/Death assay: 2X calcein AM/EthD-III working solution was prepared by adding calcein AM and EthD-III stock solutions to PBS to a concentration of 6 μM. 100 μL of 2X working fluid was added to each well for a final volume of 200 μL and a final concentration of 3 μM for each dye. The plates were incubated for 1 hour at room temperature, protected from light. Fluorescence was detected on a SpectraMax i3x plate reader using the settings as in Table 1. Caspase-3/7 R110 assay: The substrate assay buffer was prepared by adding the Enzyme substrate (AC-DEVD) 2-R110 (2 mM) to the cell lysis/assay buffer and mixing at a ratio of 50 μL to 1 mL. 100 μL of substrate assay buffer was added to each well to form a final volume of 200 μL per well and a final concentration of 50 μM. The samples were then incubated for 1 hour at room temperature in the dark. Fluorescence was detected on a SpectraMax i3x reader using the settings in Table 1. Caspase-3/7 NucView 488 assay: 10 μM 2X NucView 488 substrate working solution was mixed in PBS. 100 μL of substrate working solution was added to wells containing 100 μL of cells and medium to a final concentration of 5 μM. The cells were incubated for 1 hour at room temperature in the dark. Image acquisition was performed on a 541 nm green fluorescent channel using a SpectraMax MiniMax cytometer or on a SpectraMax i3x reader. See Table 1 for reading machine settings. result The results show that U937 cells undergo an apoptotic process in a dose- and time-dependent manner when treated with TNFα. GM-CSF treated U937 cells for 48 hours without any effect. However, it can be observed that there is a significant synergistic effect when cells are incubated with a mixture of TNFα and GM-CSF. It has been reported that GM-CSF alone requires a longer time (after 72 hours) to pass the caspase class 3 pathway relative to TNFα. Apoptosis of U937 cells was induced, and the synergistic effect of these two cytokines was observed after 24 hours. In our study, this synergy did not become apparent until after 48 hours. Please see Figure 2-5. to sum up By using three different reagents to detect different data on apoptosis, we clarified that the ready-to-read experimental template can simplify the assay of suspension cells. The pore-scanning function of the SpectraMax i3x reader reduces the difference between the well and the well and corrects the problem of uneven growth and distribution of the whole well. The Cell Viability Detection Reagent family provides researchers with a wide range of powerful tools for detecting cellular events such as apoptosis and/or cell death. Okuma E., Saeki K., Shimura M., Ishizaka Y., Yasugi E., Yuo A. Induction of apoptosis in human hematopoietic U937 cells by granulocyte-macrophage colony-stimulating factor: possible existence of caspase 3-like pathway. Leukemia (2000) 14, 612-619. Creatine,Carnosine, Inositol ,BCAA ,Glutamine PYSON Co. ,Ltd. , https://www.pysonbio.com
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