Journal: Journal of Cell Communication and Signaling

Article Title: Iron enhances reactive oxygen species generation and initiates neutrophil extracellular traps formation on the endothelium to exacerbate stroke

doi: 10.1002/ccs3.70050

Figure Lengend Snippet: NETs are induced on ECs under H/R, dependent on ROS and iron. ECs were labeled with PKH26 (red) before reoxygenation, NETs were visualized using SYTOX Green, and SYTOX Green‐positive areas were analyzed using ImageJ. (A) The work flow of induction NETs and treatment in vitro. (B) MAECs ( N = 6), HUVECs ( N = 9), and mouse brain angioendothelioma (bEnd.3, N = 9) cells subjected to H/R were incubated with neutrophils to induce NETs. EDTA, DFOM, and Vc were co‐administered with neutrophils. (C) Mouse neutrophils subjected to H/R were incubated with normal MAECs to assess NETs formation. (D) NETs formation was confirmed by assessing CitH3 protein expression in both MAECs and neutrophils after H/R, N = 3. (E) NETs formation was confirmed in bEnd.3 cells and neutrophils after H/R, N = 3. p < 0.05 was considered statistically significant, as determined by one‐way ANOVA followed by Tukey's multiple comparisons test. DFOM, deferoxamine mesylate; ECs, endothelial cells; EDTA, ethylenediaminetetraacetic acid; H/R, hypoxia/reoxygenation; HUVECs, human umbilical vein ECs; MAECs, mouse aortic endothelial cells; NETs, neutrophil extracellular traps; ROS, reactive oxygen species; Vc, vitamin C.

Article Snippet: Then the culture medium was changed to 2% FBS, and the ECs were stained with PKH26 Red Fluorescent Cell Linker (5 μM, HY‐D1451, MCE).

Techniques: Labeling, In Vitro, Incubation, Expressing

Journal: bioRxiv

Article Title: Type-I interferon priming signal balances anti-bacterial and anti-tumor trained immunity in alveolar macrophages

doi: 10.1101/2025.06.14.659659

Figure Lengend Snippet: (A) Representative flow cytometry histograms of surface molecules on peripheral blood monocytes and AMs from uninfected (PBS) or IAV-infected WT or IFNAR -/- mice. (B) Heatmaps of RNA-seq data showing genes transcripts of core transcriptional factors (TFs) of AMs, embryonic-derived and monocyte-derived signature genes in AMs. (C) ATAC-seq peaks of Marco , Car4 , and Rxra in AMs. (D) Schema of PKH26 staining of AMs before IAV infection in WT or IFNAR -/- mice. (E) Percentage of PKH26 + cells in peripheral blood monocytes and AMs immediately before IAV infection. (F) Representative flow cytometry histograms of PKH26 fluorescence in AMs from BAL and digested lung tissues. (G) Percentages of PKH26 + AMs in BAL and digested lung tissues. (H) Concentrations of representative proinflammatory cyto-/chemokines, including TNF, IL-6, and MIP-2, in supernatants of ex vivo stimulated PKH26 + AMs purified from IAV-infected or uninfected (PBS) WT mice. (I) Schema of parabiotic mouse model. (J,K) Representative flow cytometry dot plots showing the chimerisms of circulating monocytes (J) and AMs isolated from BAL and digested lung tissues (K) in parabiotic mice as shown in I . (L) Schema of IAV or S.P. infection in an AM fate-mapping Cx3cr1-YFP CreER R26 tdTomato mouse model. (M,N) Representative flow cytometry dot plots showing tdTomato fluorescence in AMs (M) and percentages of tdTomato + AMs (N) from uninfected (PBS), IAV-infected, or S.P. infected mice at 30 days post infection as shown in L . Bar graphs are presented as mean ± SD. Data in A , D - K are representatives of three independent experiments with n = 2, 3, or 4 mice or duplicated culture wells per group. Data in M and N are representatives of two independent experiments with n = 2, 4, or 5 mice per group. Data in B and C are from one experiment with n = 3 mice per group. Numbers in J , K , and M indicate percentage against parent gate. Two-tailed Student t test was performed for comparisons between two groups. One-way ANOVA followed by a Tukey test was performed to compare more than two groups.

Article Snippet: Briefly, PKH26 cell labeling kit (MCE) was prepared according to manufacturer’s recommendation and a single dose of PKH26 was administered intranasally into the lungs of mice (10 μM, 50 μL per mouse) at 5 days before IAV infection.

Techniques: Flow Cytometry, Infection, RNA Sequencing, Derivative Assay, Staining, Fluorescence, Ex Vivo, Purification, Isolation, Two Tailed Test