Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Fluorescence microscopy of representative control (B6/J, Ccr2 RFP+/+ Cx3cr1 GFP+/+ , and R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) and SAU (R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) mouse retinas for RFP and GFP at 50 days of age. 40x magnification.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Fluorescence, Microscopy, Control

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Flow cytometry distinguishes retinal MG from infiltrating macrophages. (A, B) Analysis of peripheral blood mononuclear cells for RFP and GFP expression showing the background levels of RFP and GFP fluorescence in B6/J mice (A) and the presence of RFP + , GFP + and RFP + GFP + cells in the circulation of Ccr2 RFP+/+ Cx3cr1 GFP+/+ mice (B) . The gating strategy for selecting CD45 + cells from B6/J mice is the same as shown for Ccr2 RFP+/+ Cx3cr1 GFP+/+ mice. (C) Representative analysis of a B6/J mouse showing background levels of RFP and GFP fluorescence in retinal CD45 + cells. Background fluorescence for GFP indicated as GFP neg cells. (D) Representative analysis of R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas showing that a minimal number of all CD45 + cells (gate 1) express Ccr2 (RFP), and that a single population comprising most of the CD45 + cells and specifically the CD45 med cells (gate 2) express a high level of Cx3cr1 (GFP). Retinal CD45 + cells negative for GFP expression (GFP neg ) or with a low or intermediated level of GFP expression (GFP lo-med ) are indicated. (E) Representative analysis of R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas with SAU showing a large increase in both CD45 med and CD45 hi cells with most of the RFP + cells found in the CD45 hi population as either RFP + GFP neg or RFP + GFP lo-med cells and that they are myeloid cells or lymphocytes. For all analyses; retinal MG indicated by red circles, flow cytometry done at 50 days of age, n = 4 for all groups of mice, and the mean percentage of cells in each quadrant for all mice analyzed is indicated.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Flow Cytometry, Analysis, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Analysis of R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas for Ccr2 + (RFP + ) cells following ONC. (A) Representative fundoscopy of pre-ONC and day 10 post-ONC retinas. (B) Representative flow cytometry analysis of retinal CD45 med cells for RFP and GFP at day 10 post-ONC with mean percentage of CD45 med cells expressing GFP and RFP from all examined retinas indicated.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Analysis, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Selenoprotein P Deficiency Drives Hepatocellular Carcinoma Progression via Induction of Neutrophil Senescence and Immunosuppressive Microenvironment

doi: 10.1101/2025.06.24.661430

Figure Lengend Snippet: (a) Representative images of livers from S100a8-Cre and Lrp8 f/f ; S100a8-Cre ( Lrp8 ΔNeu ) mice at endpoint, demonstrating increased tumor burden in Lrp8 ΔNeu mice. (b, c) Quantification of liver-to-body weight ratio (b) and tumor counts (c) in Lrp8 ΔNeu versus control groups, showing a significantly higher tumor burden in the Lrp8 ΔNeu group (n = 9 per group). (d) Flow cytometry analysis showing an increased percentage of neutrophils in the tumor microenvironment of Lrp8 ΔNeu mice compared to controls (n = 6 per group). (e, f) Flow cytometric analysis of senescence markers in tumor-infiltrating neutrophils from represent mice. Senescence-associated β-galactosidase (SA-β-Gal) positive neutrophils (e) and p21 + neutrophils (f) were significantly increased in Lrp8 ΔNeu mice compared to control group (n = 5-6 per group). (g, h) Flow cytometric analysis of neutrophils isolated from Lrp8 ΔNeu and control tumors. Representative histogram showing NIR-H 2 SE fluorescence, a hydrogen selenide probe, in neutrophils. Quantification of the percentage of NIR-H 2 SE + neutrophils (g) and mean fluorescence intensity (MFI) of NIR-H 2 SE in neutrophils (h), showing significantly reduced hydrogen selenide levels in neutrophils after Lrp8 knockout (n = 5–6 per group). (i) Quantitative RT-PCR analysis of senescence-associated markers Cdkn1a in tumor-infiltrating neutrophils (n = 5–6 per group). (j, k) mRNA expression levels of S100a8 and S100a9 significantly elevated in tumor-infiltrating neutrophils from Lrp8 ΔNeu mice (n = 5-6 per group). (l) Schematic representation of hydrodynamic tail vein injection (HDTVi) constructs. Mice were injected with constructs encoding oncogenic NRas G12V and myr-AKT along with a plasmid that encodes a green fluorescent protein (GFP) -Sepp1 fusion protein for tracking Sepp1 localization. (m) Representative immunofluorescence images of liver tissues showing GFP (green) indicating Sepp1 localization, S100A9 (red) indicating neutrophils, lymphocyte antigen 6 family member G (Ly6G; white) as another neutrophil marker, and 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Images demonstrate the colocalization of GFP with neutrophils, indicating that neutrophils uptake Sepp1. The lower row of images shows magnified views of the boxed regions, highlighting Sepp1 uptake by neutrophils. Data are presented as mean ± SEM; statistical significance was determined using two-tailed unpaired Student’s t-tests.

Article Snippet: Tumor sections were blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature, after which sections were incubated (overnight at 4 °C) in PBS-1% BSA containing a primary antibody against green fluorescent protein (GFP; 1:500, Abmart, Cat. M20004), Ly6G (1:200, Servicebio, Cat. GB11229), or S100A9 (1:500, R&D Systems, Cat. AF2065).

Techniques: Control, Flow Cytometry, Isolation, Fluorescence, Knock-Out, Quantitative RT-PCR, Expressing, Injection, Construct, Plasmid Preparation, Immunofluorescence, Marker, Two Tailed Test