Journal: Communications Biology

Article Title: NUPR1 protects against hyperPARylation-dependent cell death

doi: 10.1038/s42003-022-03705-1

Figure Lengend Snippet: a OXPHOS metabolism, reflected by oxygen consumption rate (OCR) levels were measured in MiaPaCa-2 cells after 24 h treatments. b MiaPaCa-2 cells were treated with ZZW-115 (1.5 µM) and olaparib (25 µM) for 24 h, then, loaded with MitoTracker Deep-Red FM and, after fixation, stained with DAPI. Flow cytometry analysis were carried out using MitoProbe™ TMRM ( c ), MitoSOX™ Red ( d ), CellROX™ Orange Reagent ( e ) or Fluo-4-AM ( f ) for analysis of the mitochondrial membrane potential, mitochondrial superoxide levels, intracellular ROS levels and the cytosolic calcium concentration, respectively, after 24 h of incubation with the drugs. Data represent mean ± SEM. Two-way, Sidak was used, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001. g PLA was performed in MiaPaCa-2 cells in the presence or in the absence of ZZW-115 together or not with 5-FU in the presence or absence of olaparib. Mouse anti-PAR and rabbit anti-Mitofusin2 antibodies were used. A representative experiment is shown ( n = 3). ImageJ was used to count the number of green dots. Mean ± SEM of foci/nucleus are included.

Article Snippet: Finally, samples were mounted using the Prolong Gold antifade reagent with DAPI (Thermon Fisher).

Techniques: Staining, Flow Cytometry, Membrane, Concentration Assay, Incubation

Journal: Blood. Red cells & iron

Article Title: Age-dependent neuroinflammatory effects of red blood cells and their exosomes in a human brain-on-chip model

doi: 10.1016/j.brci.2025.100044

Figure Lengend Snippet: (A) Schematic of the microfluidic device revealing 3 layers of PMMA sheets used for fabrication. The bottom layer incorporates gold electrodes for TEER measurement; the middle layer has 3 interconnected microchannels; and the top layer has inlets and outlets. (B) Image of the microfluidic device used in this study. (C) Integration of the 4 hB-MPS devices into a scalable platform and TEER measurements. (D) Integration of scalable hB-MPS platform to an electrical impedance spectroscopy system to provide output from multiple chips. (E) Quality control of the hB-MPS microfluidic devices measuring TEER of 1× phosphate-buffered saline buffer using electrical impedance spectroscopy. This resistance was used to quality control the reproducibility of the chips before performing experiments. (F) Schematic illustration of cell seeding in different microchannels of hB-MPS. (G) Confocal micrograph of hB-MPS revealing the endothelial monolayer (ZO1, red), astrocytes (GFAP, green), microglia (CD68, yellow), and coculture of astrocytes and microglia (GFAP, green; CD68, yellow; and DAPI, blue) cultured under fluid flow conditions for 5 days. (H) TEER measurements of the barrier formation collected in 5 days. Increased resistance was observed on day 5, when a complete barrier was formed. GFAP, glial fibrillary acidic protein. Panel F created with biorender.com . Mehta-Doshi. (2025)

Article Snippet: ProLong Gold Antifade with DAPI (4′, 6-diamidino-2-phenylindole; LifeTech) was used to label nuclei.

Techniques: Impedance Spectroscopy, Control, Saline, Cell Culture

Journal: Blood. Red cells & iron

Article Title: Age-dependent neuroinflammatory effects of red blood cells and their exosomes in a human brain-on-chip model

doi: 10.1016/j.brci.2025.100044

Figure Lengend Snippet: (A) Representative confocal micrographs revealing microglia (CD68, yellow; DAPI, blue) exposed to RBCs of young and old healthy donors (male and female). Scale bar, 50 μm. Accumulation of heme was determined using anti–HO-1 antibody (red). Scale bar, 25 μm. The images were obtained using a Leica SP8 confocal microscope with lighting deconvolution. (B) Quantification of CD68 protein levels in the microglial cells based on fluorescence intensity. (C) Quantification of heme levels in the brain tissue based on fluorescence intensity. The CD68 and HO-1 fluorescence intensity data are normalized to control and presented as mean ± standard error of the mean (SEM; n = 3). One-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons was performed to determine the significance across different groups. * P < .05; ** P < .01; *** P < .0001. CFTI, corrected fluorescence total intensity; HO-1, heme oxygenase 1; ns, not significant; OF, old female; OM, old male; YF, young female; YM, young male.

Article Snippet: ProLong Gold Antifade with DAPI (4′, 6-diamidino-2-phenylindole; LifeTech) was used to label nuclei.

Techniques: Microscopy, Fluorescence, Control

Journal: Blood. Red cells & iron

Article Title: Age-dependent neuroinflammatory effects of red blood cells and their exosomes in a human brain-on-chip model

doi: 10.1016/j.brci.2025.100044

Figure Lengend Snippet: (A) Representative graph showing mean diameter of exosomes extracted from the RBCs of a YM donor. The mean diameter was 161.39 nm. (B) Representative graph showing mean diameter of exosomes extracted from the RBCs of an OM donor. The mean diameter was 194.16 nm. (C) Mean diameter of the exosomes from the RBCs of young and old donors (n = 3; mean ± SEM) using nanoparticle tracking analysis. (D) Validation of exosomes abundance using enzyme-linked immunosorbent assay for CD63 surface marker. Concentration of the exosomes (particles per milliliter) derived from the RBCs of young and old donors (n = 3; mean ± standard deviation [SD]). (E) Confocal micrographs of microglia (CD68, yellow; DAPI, blue) exposed to RBC-derived exosomes of young and old healthy donors (male and female). (F) Quantification of CD68 levels in microglial cells exposed to the RBC-derived exosomes in the vascular microchannel of the hB-MPS. The images were obtained using Leica SP8 confocal microscope with lightning deconvolution. Quantification of CD68 intensities using image analysis was normalized to control and presented as mean ± SEM (n = 3). One-way ANOVA followed by Tukey test for multiple comparisons was performed to compare significance across different groups. * P < .05; ** P < .01; *** P < .0001. CFTI, corrected fluorescence total intensity; OF, old female; OM, old male; PSD, particle size distribution; YF, young female; YM, young male.

Article Snippet: ProLong Gold Antifade with DAPI (4′, 6-diamidino-2-phenylindole; LifeTech) was used to label nuclei.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Marker, Concentration Assay, Derivative Assay, Standard Deviation, Microscopy, Control, Fluorescence