Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Detecting Purinosome Metabolon Formation with Fluorescence Microscopy

doi: 10.1007/978-1-4939-7759-8_17

Figure Lengend Snippet: Colocalization of endogenous FGAMS and GART for visualization of purinosomes by immunofluorescence. Purine-depleted HeLa cells were fixed and permeabilized prior to being probed for with FGAMS rabbit polyclonal antibody and GART mouse monoclonal antibody. Fluorescently labeled secondary antibodies CF488A-conjugated donkey anti-rabbit and CF568-conjugated donkey anti-mouse were used to visualize the expression and localization of FGAMS and GART, respectively. A representative image of an individual cell was captured using a 100× oil objective on an Olympus Fluoview 1000 confocal laser scanning microscope. Sequential imaging of CF488A and CF568 showed colocalization of (a) FGAMS with (b) GART as represented by the yellow puncta present in (c) the merged image. Scale bar: 10 μm

Article Snippet: Primary antibody solution: 1:500 dilution of PFAS rabbit polyclonal antibody (Bethyl Laboratories) and 1:1000 dilution of GART mouse monoclonal antibody (Novus Biologicals) prepared in blocking buffer.

Techniques: Immunofluorescence, Labeling, Expressing, Laser-Scanning Microscopy, Imaging

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