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Ganglioside GD2 Specific Antibodies in the Diagnosis and Therapy of Human Neuroblastoma
Published in John T. Kemshead, Pediatric Tumors: Immunological and Molecular Markers, 2020
Nai-Kong V. Cheung, Floro D. Miraldi
Both the IgM and the IgG3 MAb to GD2 activate human complement efficiently in tumor cytotoxicity.20,25 Using the Hoechst stain method20 to monitor the rare tumor cells, as many 10% tumor cells in the bone marrow can be eliminated without damaging normal marrow stem cells. Normal human cells are resistant to complement lysis because of the presence of decay-accelerating factor (DAF) on their cell surface.16 However, neuroblastomas and many melanomas have low to absent expression of this protein and, therefore, are very sensitive to human complement. This sensitivity of human neuroblastoma cells to complement has important therapeutic implications. With the activation of human complement, anaphylatoxic and chemotactic properties of activated complement fragments can play an important role in the formation of local inflammatory response as well as the influx of important effector cells to tumor sites. Complement receptors for C3b and C3bi are present on granulocytes and natural killer cells. Since C3b and C3bi are deposited on tumor cells after MAb activation of human complement, they may enhance such cell mediated tumor cytotoxicity.
In Vitro Anticancer Activity of Syzygium calophyllifolium on A549 Lung Cancer Cells
Published in Parimelazhagan Thangaraj, Phytomedicine, 2020
Rahul Chandran, Heidi Abrahamse
The damage caused by the SCBM extract was analyzed using Hoechst nuclear stain. The MCF-7 cells were cultured in 3.4 cm diameter culture dishes over sterile cover slips and allowed to reach a confluence of 80%. The cells were then treated with different concentrations of SCBM (5, 10, and 20 μg). After 24 hours of incubation, the cells were stained with 1 μg/mL Hoechst stain (Hoechst 33258, H21491) for 30 minutes. Thereafter, the cells were rinsed with Phosphate Buffer Saline (PBS), and the blue fluorescent signal was examined using the Olympus fluorescent microscope.
The unforeseen intracellular lifestyle of Enterococcus faecalis in hepatocytes
Published in Gut Microbes, 2022
Natalia Nunez, Aurélie Derré-Bobillot, Nicolas Trainel, Goran Lakisic, Alexandre Lecomte, Françoise Mercier-Nomé, Anne-Marie Cassard, Hélène Bierne, Pascale Serror, Cristel Archambaud
The Huh7 hepatocytes were seeded in a cell culture µ-dish (Clinisciences, ibidi 81156) 4 d before infection and then infected with GFP-expressing OG1RF E. faecalis as described in the section on cell infection. After 12 h of infection, 1 mM orange-red TAMRA-based fluorescent D-amino acid (RADA, Tocris, 6649) was added to the µ-dish. After 38 h of infection, cells were washed 5 times in Hanks’ balanced salt solution (HBSS) 1X and fixed in 4% PFA for 20 min at room temperature. The Hoechst stain (Sigma B2261, 5 µg/ml) was used to stain DNA. As a control, remaining antibiotic-killed extracellular enterococci were detected using the rabbit anti-Enterococcus antiserum (diluted 1:1000) and a goat anti-rabbit-Alexa Fluor 647-conjugated secondary antibody (ThermoFisher Scientific, A-21244 diluted 1:200), as described above.
Amomum subulatum Induces Apoptosis in Tumor Cells and Reduces Tumor Burden in Experimental Animals via Modulating Pro-Inflammatory Cytokines
Published in Cancer Investigation, 2021
Drishya Sudarsanan, Dhanisha Suresh Sulekha, Guruvayoorappan Chandrasekharan
DLA cells (1 × 106 cells) were freshly aspirated from mice peritoneal cavity and washed in PBS (pH 7.4). Cells were incubated with MEAS (400 µg/ml) for 3 h. For AO/EB staining assay, the cell suspension was mixed with AO/EB dye (1 µl; 100 µg/ml) on clean microscopic slides and observed under Olympus BX43 microscope, Olympus Life Science Solutions (Shinjuku, TYO, Japan) using FITC filter (20). For Hoechst 33342 staining assay, the cell suspension was mixed with Hoechst stain 33342 stain (5 µl; 1 mg/ml) and incubated for 20 min. The cells were then observed under Olympus BX43 microscope, Olympus Life Science Solutions, using blue filter (21). For H and E staining, the cell suspension was fixed on a clean microscopic slide with 10% formalin and staining steps were carried out as per the method of Fischer et al. (22) with slight modifications. Briefly, fixed slides were washed twice in PBS (pH 7.4) and immersed in Harris hematoxylin for about 2 min. Slides were further washed with water for 1 min (twice) and immersed in 0.3% ammonium hydroxide solution (10–20 dips). After washing, slides were immersed in eosin solution (10 dips) and further dehydrated with increasing concentrations of ethanol. Slides were observed under Olympus CKX53 microscope, Olympus Life Science Solutions
Development of Lactobacillus kimchicus DCY51T-mediated gold nanoparticles for delivery of ginsenoside compound K: in vitro photothermal effects and apoptosis detection in cancer cells
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Yeon-Ju Kim, Haribalan Perumalsamy, Josua Markus, Sri Renukadevi Balusamy, Chao Wang, Seong Ho Kang, Seungah Lee, Sang Yong Park, Sung Kim, Verónica Castro-Aceituno, Seung Hyun Kim, Deok Chun Yang
RAW264.7, AGS, A549 and HT29 cells were seeded onto glass coverslips and incubated in six-well plates at a density of 2.5 × 105 cells/mL for 24 h at 37 °C in a humidified incubator with 5% CO2 and 95% air. The cells were then incubated with a suspension of DCY51T-AuNps or DCY51T-AuCKNps (1 or 5 µg/mL; equivalent to 0.18 and 0.89 µM of ginsenoside CK) for 24 h. Next, the cells were washed with 1× PBS to remove excess nanoparticles and fixed with 3.7% (v/v) formaldehyde for 5 min at room temperature. The cells were further incubated in fresh culture medium for 24 h. Subsequently, RAW264.7, A649 and HT29 cells were irradiated with a 635 nm laser (Shanghai Dream Laser Technology Co. Ltd., Shanghai, China) with a heat flux density of 0.74 W/cm2 directly above the cell plates for 10 min with a laser spot size of 0.2 cm2, resulting in a total energy dose of 88.80 J. On the other hand, AGS cells were irradiated with an 800 nm laser. After irradiation, the cells were maintained at 37 °C in a humidified incubator with 5% CO2. Finally, the irradiated cells were stained by Hoechst 33258 solution (2 µg/mL) for 20 min in the dark at room temperature to analyse synergistic cell apoptosis [30]. Images of Hoechst stain were acquired by a fluorescence microscope (×400, Optinity, Korean Labtech, Namyangju, South Korea). In all studies, cells without nanoparticles and cells incubated with DCY51T-AuNps at 5 µg/mL served as blank and negative control and were subjected to the same irradiation conditions as cells incubated with DCY51T-AuCKNps.