Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: PeerJ

Article Title: Cirsilineol improves anesthesia/surgery-induced postoperative cognitive dysfunction through attenuating oxidative stress and modulating microglia M1/M2 polarization

doi: 10.7717/peerj.18507

Figure Lengend Snippet: Sequences of primers.

Article Snippet: Following washing with PBS, the brain tissues and BV-2 cells were incubated with the blocking buffer at 4 °C for 1 h and the primary antibodies against Iba-1 (019-19741; 1:1,000, Wako Fujifilm, Osaka, Japan), CD86 (942-RBM-4-P1; 1:1,000, Thermo Fisher Scientific) and CD206 (CL594-60143; 1:100, Proteintech, Wuhan, China) at 4 °C overnight.

Techniques: Sequencing

Journal: PeerJ

Article Title: Cirsilineol improves anesthesia/surgery-induced postoperative cognitive dysfunction through attenuating oxidative stress and modulating microglia M1/M2 polarization

doi: 10.7717/peerj.18507

Figure Lengend Snippet: (A and B) Iba-1 + cells in each group of mice based on the quantification on the fluorescence intensity in immunofluorescence assay. (C and D) The calculated levels of microglia polarization indicators Cd86 (M1) and Cd206 (M2). (E and F) The gauged levels of SOD and MDA in plasm of mice in each group. Results were expressed as mean ± standard deviation. ns represents non-significant; P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Following washing with PBS, the brain tissues and BV-2 cells were incubated with the blocking buffer at 4 °C for 1 h and the primary antibodies against Iba-1 (019-19741; 1:1,000, Wako Fujifilm, Osaka, Japan), CD86 (942-RBM-4-P1; 1:1,000, Thermo Fisher Scientific) and CD206 (CL594-60143; 1:100, Proteintech, Wuhan, China) at 4 °C overnight.

Techniques: Fluorescence, Immunofluorescence, Standard Deviation

Journal: PeerJ

Article Title: Cirsilineol improves anesthesia/surgery-induced postoperative cognitive dysfunction through attenuating oxidative stress and modulating microglia M1/M2 polarization

doi: 10.7717/peerj.18507

Figure Lengend Snippet: (A–D) Quantified CD86 MFI (A, C) and CD206 MFI (B, D) in each group based on immunofluorescence assay. (E, F) The gauged levels of SOD and MDA in the supernatant of cell culture medium in each group. Results were expressed as mean ± standard deviation. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Following washing with PBS, the brain tissues and BV-2 cells were incubated with the blocking buffer at 4 °C for 1 h and the primary antibodies against Iba-1 (019-19741; 1:1,000, Wako Fujifilm, Osaka, Japan), CD86 (942-RBM-4-P1; 1:1,000, Thermo Fisher Scientific) and CD206 (CL594-60143; 1:100, Proteintech, Wuhan, China) at 4 °C overnight.

Techniques: Immunofluorescence, Cell Culture, Standard Deviation

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: RBC-MBs promotes the polarization of macrophages towards the M2 phenotype by inhibiting the TLR9-MyD88 and cGAS-STING pathways (A-F) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of pro-inflammatory cytokines (A-B), key proteins in the TLR9-MyD88 (C-D) and cGAS-STING (E-F) pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, and then subjected to western blot analysis (A, C, E). IL-1α, TNF-α, IL-6, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample. p-NF-κB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis (B, D, F), n = 6. (G) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of CD86 in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (left panel) and quantitative fluorescence graph (right panel) are shown. Scale bar: 50 μm, n = 6. (H-J) RBC-MB treatment significantly inhibited hepatic IRI-induced polarization of macrophages toward the M1 phenotype (H) and activation of the TLR9-MyD88 and cGAS-STING pathways (I-J). MASLD rats underwent 45 min of hepatic ischemia followed by reperfusion. At 0, 6, 12, 18, and 24 h post-reperfusion, the rats were treated with either Con-MBs or RBC-MBs. Primary macrophages were isolated from rat livers, then co-stained with CD68 and CD163 followed by flow cytometric analysis (H), n = 3/4, or subjected to western blotting (I-J). CD206, iNOS, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample, and these normalized values were used for statistical analysis (J), n = 3. For (B, D, F, G right panel , H right panel , J), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (B, D, F, G right panel ) or one-way analysis of variance (ANOVA) (H right panel , J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Incubation, Concentration Assay, Western Blot, Fluorescence, Microscopy, Activation Assay, Isolation, Staining, Standard Deviation

Journal: Aging Cell

Article Title: Telomerase Knockout in Myeloid Cells Predisposes Mice to Foam Cell Formation, Dyslipidemia, Lung Fibrosis, and Cardiac Dysfunction

doi: 10.1111/acel.70490

Figure Lengend Snippet: Tert KO induces lipid‐associated macrophages (LAMs). Starch‐induced i.p. macrophages from 2‐year‐old female mice were analyzed. (a) Upon adherence in primary culture, IF with antibodies against CD80 (M1‐macrophage) and CD206 (M2‐polarization) markers reveals a lower frequency of CD206 + macrophages in KO mice. (b) Data quantification from multiple fields of view in (a), indicating macrophage polarization shift toward the M1 phenotype. (c) Upon LPS (100 ng/mL, 4 h) treatment in primary culture, q‐RT‐PCR (normalized to 18S RNA) demonstrates higher expression of genes coding for inflammation markers IL1 and IL6 in KO macrophages. (d) Macrophages were induced to convert into foam cells by oxLDL (0.025 mg/mL) treatment for 24 h. Note increased uptake of red‐fluorescent C 12 ‐BODIPY (0.3 μM, 5 min) by mG+ KO cells (yellow arrows) compared to mG+ WT cells (green arrows) in primary culture. (e) q‐RT‐PCR (normalized to 18S RNA) demonstrates lower expression of genes coding for lipid efflux effectors APOE, LDLR, ABCA1, ABCG1, and higher expression of genes coding for lipid transporters CD36 and FABP5 in KO oxLDL‐treated macrophages. (f) OxLDL‐treated macrophages stained with Oil Red O: Note larger lipid droplets (arrows) in KO cells. For all data, mean+/− SEM (error bars). * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐sided Student's t ‐test). Scale bar: 50 μm.

Article Snippet: For immunofluorescence (IF), the following antibodies were used: CD206 (R&D Systems, AF2534, 1:75); CD80 (Abclonal A23688, 1:50); CD68 (Invitrogen, MA5‐1324, 1:75); F4/80 (Abcam, ab16911, 1:50); TERT (Biossusa, bs‐1411R, 1:50).

Techniques: Starch, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining

Journal: Aging Cell

Article Title: Telomerase Knockout in Myeloid Cells Predisposes Mice to Foam Cell Formation, Dyslipidemia, Lung Fibrosis, and Cardiac Dysfunction

doi: 10.1111/acel.70490

Figure Lengend Snippet: AT abnormalities in LysM‐ Tert KO mice. (a) Senescence‐associated β‐galactosidase staining of VAT from 20‐month‐old female mice. (b) Senescence‐associated β‐galactosidase staining of adherent cells from VAT in (a). Arrows: Senescent cells. (c) Flow cytometry on VAT from A, revealing a lower frequency of mG+ macrophages expressing CD206 in KO mice. (d) Flow cytometry on SAT, revealing a higher frequency of mG+ macrophages expressing CD86 in KO mice. (e) IF with antibodies against CD68 and CD206 reveals a lower frequency of CD206+ macrophages (red arrows) in SAT of KO mice. IF with antibodies against perilipin‐1 and F4/80 reveals comparable adipocyte size in SAT of WT and KO mice. (f) Trichrome staining reveals fibrosis (arrows) in SAT of KO mice. In (d, e) 4‐month‐old male mice fed an atherogenic diet were used. Scale bar: 50 μm.

Article Snippet: For immunofluorescence (IF), the following antibodies were used: CD206 (R&D Systems, AF2534, 1:75); CD80 (Abclonal A23688, 1:50); CD68 (Invitrogen, MA5‐1324, 1:75); F4/80 (Abcam, ab16911, 1:50); TERT (Biossusa, bs‐1411R, 1:50).

Techniques: Staining, Flow Cytometry, Expressing

Journal: Aging Cell

Article Title: Telomerase Knockout in Myeloid Cells Predisposes Mice to Foam Cell Formation, Dyslipidemia, Lung Fibrosis, and Cardiac Dysfunction

doi: 10.1111/acel.70490

Figure Lengend Snippet: Lung abnormalities in LysM‐ Tert KO mice. (a) IF with antibodies against F4/80 and CD206 reveals a lower frequency of CD206+ macrophages (red arrows) in lungs of 6 month‐old chow‐fed KO mice, compared to WT mice. (b) Data quantification for 10 view fields from A. * p < 0.05 (two‐sided Student's t ‐test). (c) Trichrome staining reveals fibrosis (arrows) in 20‐month‐old lungs of KO mice. (d) q‐RT‐PCR (normalized to 18S RNA) demonstrates higher expression of Tgfb1 and Cola1a in lungs of KO male and female mice. Shown are mean+/− SEM (error bars). * p < 0.0001 (two‐sided Student's t ‐test). ** p < 0.01. In (a), (b), and (d), 6‐month‐old male mice fed a chow diet were used. Scale bar: 50 μm.

Article Snippet: For immunofluorescence (IF), the following antibodies were used: CD206 (R&D Systems, AF2534, 1:75); CD80 (Abclonal A23688, 1:50); CD68 (Invitrogen, MA5‐1324, 1:75); F4/80 (Abcam, ab16911, 1:50); TERT (Biossusa, bs‐1411R, 1:50).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing