Journal: Analytical chemistry

Article Title: A Novel Omniphobic Platform for Multicellular Spheroid Generation, Drug Screening, and On-Plate Analysis

doi: 10.1021/acs.analchem.1c01326

Figure Lengend Snippet: A) Representative confocal image of live (Calcein AM, green) and dead (Ethidium Homodimer, red) cells within 1000 OVCAR8 cells/20 μL spheroids generated on an omniphobic mesh plate, imaged on day 7. B) Quantification of live and dead cells within spheroids generated on the omniphobic mesh plate, from confocal images (two-tailed t-test, n>3, *p<0.0002). C) Representative phase images of spheroids on the omniphobic mesh plate (OMP) and 384-hanging drop plate (384-HDP) after 48 hours treatment with 10 μM of the anti-cancer drug paclitaxel. D) Quantification of percent viable cells within spheroids after paclitaxel treatment, as analyzed by resorufin fluorescence. The viability was normalized to control treatment with cell culture medium (one-way ANOVA, n>3, *p<0.0001). The omniphobic mesh plate demonstrated equivalent spheroid viability on the first (OMP-1) and fourth (OMP-4) use of a plate. E) Representative confocal microscope images of heterogeneous spheroids generated with 1:1 ratio of mTomato labeled Pt224 ovarian cancer stem cells (500 cells, CD133+ and ALDH+) and GFP labeled human embryonic kidney (HEK) cells (500 cells) on day 5. F) Graph representing viability of 1000 cells spheroids generated using OVCAR8 and OVCAR3-GFP cell lines, in response to 24 hr of chemotherapeutic drug (20 µM doxorubicin, 10 µM paclitaxel) across three different spheroid generating platforms: omniphobic mesh (OMP), 384 hanging drop plate (384-HDP) and commercially available, 96 well U-bottom plate (U-bottom). A significant difference in viability was observed (two way ANOVA, n=4, ****p< 0.0001) when comparing no treatment control to Doxorubicin and Paclitaxel across all platforms. Additionally, a significant difference was observed in (two way ANOVA, n=4, **** p<0.0001) OVCAR3-GFP OMP & HDP, compared to OVCAR3 U-bottom, (two way ANOVA, n=4, **** p<0.0001), and in OVCAR8 OMP compared to OVCAR8 U-bottom. There was also significant change (two way ANOVA, n=4, *** p<0.0008) OVCAR8 HDP compared to OVCAR8 U-bottom.

Article Snippet: The stained cells were carefully transferred to glass coverslips and imaged using a laser scanning confocal microscope (Olympus IX81, equipped with a Yokogawa CSU-X1 confocal scanning laser unit, Andor iXon x3 CCD camera, and Metamorph 7.8 software), with constant gain and exposure settings between samples.

Techniques: Generated, Two Tailed Test, Fluorescence, Control, Cell Culture, Microscopy, Labeling

Journal: European Journal of Immunology

Article Title: NET formation can occur independently of RIPK3 and MLKL signaling

doi: 10.1002/eji.201545615

Figure Lengend Snippet: The formation of human NETs occurs independently of MLKL signaling . Human blood neutrophils were isolated from healthy donors by Ficoll‐Hypaque centrifugation. (A) Confocal microscopy. NET formation following short‐term stimulation (total 45 min) of human neutrophils with the indicated triggers in the presence and absence of 5 μM NSA. The number of NET‐forming neutrophils was determined by counting the DNA‐releasing cells in ten high power fields ( n = 5). Representative original data (right). Co‐localization of elastase (green) with released DNA (PI, red) is indicated by the yellow color. Bars, 10 μM. (B) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye ( n = 3). (C) Total ROS production by activated human neutrophils in the presence and absence of 5 μM NSA was measured by flow cytometry. During cell activation, cells were incubated with 1 μM DHR123 ( n = 3). (D) Bacterial killing by colony forming unit (cfu) assay. Human neutrophils exert antibacterial activity against E. coli that can be partially blocked by 100 U/mL DNase I both in the presence and absence of 5 μM NSA ( n = 3). (E) Viability of Jurkat cells was analyzed by ethidium bromide exclusion assay. In parallel experiments, 5 μM NSA blocked cell death induced by 20 ng/mL TNF‐α plus 100 nM Smac mimetic AT‐406 (cIAP1/2 selective IAP‐antagonist) in caspase‐8 deficient and 20 μM zVAD‐FMK–treated Jurkat cells ( n = 3), demonstrating that the inhibitor was pharmacologically active. All data are shown as mean ± SEM of the indicated number of independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; one‐way ANOVA test.

Article Snippet: The monovalent Smac mimetic compound AT‐406 was from Selleckchem (Houston, TX, USA) and the ROS‐Glo TM H 2 O 2 kit was purchased from Promega AG (Dübendorf, Switzerland).

Techniques: Isolation, Centrifugation, Confocal Microscopy, Flow Cytometry, Activation Assay, Incubation, Colony-forming Unit Assay, Activity Assay, Exclusion Assay