Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 2 High SERPINE1 expression in GC cells promotes macrophage M2 polarization. tSNE visualization of nine single-cell clusters partitioned by unsu pervised cluster analysis, SERPINE1 expression of each single-cell, and SERPINE1 expression abundance of different single-cell clusters in the GSE134520 (A–C) and GSE167297 (D–F) datasets. (G) Flow cytometry analysis of the proportion of CD68+CD206+ macrophages in a Transwell co-culture system, with MKN45 and AGS cells overexpressing (oe_SERPINE1) or silencing SERPINE1 (shRNA#3 or sh_SERPINE1#3) in the upper chamber, and THP1 cells treated with PMA in the lower chamber. (H) Immunofluorescence staining of xenograft tumor tissues. Comparison of the proportion of M1 or M2 macrophage infiltra tion. Green indicates F4/80. Red indicates iNOS or Arg1 expression

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, shRNA, Immunofluorescence, Staining, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Gastric cancer-derived exosomal let-7 g-5p mediated by SERPINE1 promotes macrophage M2 polarization and gastric cancer progression.

doi: 10.1186/s13046-024-03269-4

Figure Lengend Snippet: Fig. 5 SERPINE1-mediated gastric cancer-derived exosomes facilitate the polarization of THP1 cells into M2 macrophages. (A) Schematic representation of the extraction and identification of exosomes and the induction of macrophage polarization. Transmission electron microscopy (B), nanoparticle tracking analysis (C), and western blotting (D) were used to identify the morphology, particle size, and markers of exosomes. (E) Confocal laser scanning microscopy detected Dil-labeled exosomes (red) internalized by DAPI-labeled macrophages (blue). (F–G) Immunofluorescence analysis of the proportion of CD206+ cells in THP1 cells treated with exosomes. (H–I) Flow cytometry analysis of the proportion of CD68+CD206+ cells in THP1 cells treated with exosomes. (J–K) qRT-PCR analysis of M1 markers (iNOS and TNF-α) and M2 markers (TGF-β, IL-10, and Arg-1) in THP1 cells treated with exosomes. (L–N) Transwell migration and invasion assays of GC cells (upper chamber) co-cultured with macrophages (lower chamber) ingesting exosomes

Article Snippet: THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-μm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Techniques: Derivative Assay, Extraction, Transmission Assay, Electron Microscopy, Western Blot, Confocal Laser Scanning Microscopy, Labeling, Immunofluorescence, Flow Cytometry, Quantitative RT-PCR, Migration, Cell Culture

Journal: Theranostics

Article Title: Therapeutic effects of a novel electrode for transcranial direct current stimulation in ischemic stroke mice

doi: 10.7150/thno.90779

Figure Lengend Snippet: Comparison of therapeutic effects between conventional and new HD-tDCS applications in stroke. (A) Schematic diagram of the new HD electrode and 3D simulation of the HD-tDCS application. Higher relative electric potential and enhanced spatial focality were predicted in the motor cortex using the new HD-tDCS application compared with those using the conventional HD-tDCS application. (B) Timeline for HD-tDCS application and behavioral tests. Both HD-tDCS applications were used to treat the contralesional motor cortex once daily for 5 days. (C) Corner test: The percentage of left turns was significantly decreased in the MCAO+tDCS-FN group compared with that in the MCAO and MCAO+tDCS-F groups. (D) Pole test: The total time for the MCAO+tDCS-FN group was significantly decreased compared with that of the MCAO group. (E) Bar charts and performance images of the open-field test. The time in the center zone was significantly increased in the MCAO+tDCS-FN group compared with that in the MCAO and MCAO+tDCS-F groups. (F) Schematic subject images, bar charts, and performance images for the novel object recognition test. The recognition index was significantly enhanced in the MCAO+tDCS-FN group compared with that in the MCAO group. (G) Schematic diagram, bar charts, and schematic recording configuration of the movement during the fear conditioning. The percentage of freezing in the CS was significantly enhanced in the MCAO+tDCS-FN group compared with that in the MCAO+tDCS-F group. (H) Representative brain images (-0.1 mm from the bregma) and quantification of the relative infarct volume. Borders to the healthy tissue are indicated with a dot line. The infarct volume in 40 brain sections from 1.1 mm to -1.9 mm from the bregma was significantly reduced in the MCAO+tDCS-FN group compared with that in the MCAO+tDCS-F group. (I) Photomicrograph and bar chart showing the number of Iba1- and CD68-positive cells in the peri-infarct region. The number of Iba1-positive cells was significantly reduced in the MCAO+tDCS-FN group compared with that in the MCAO+tDCS-F group. All behavioral, morphological, and immunofluorescence analyses (n=5/group). Data are presented as means±SEMs. * P <0.05 and *** P <0.001 vs. each group using one-way analysis of variance with Tukey's test. IL, ipsilesional site; CL, contralesional site; MCAO, middle cerebral artery occlusion; HD-tDCS, high-definition transcranial direct current stimulation; F, conventional electrode; FN, new needle-conjugated electrode; BL, baseline; CS, cue stimulation; TI, trace interval. Magnification: ×400; scale bar=100 μm.

Article Snippet: They were then incubated overnight with the following primary antibodies (diluted in antibody dilution buffer, 1× PBS/1% bovine serum albumin/0.3% Triton X-100) at 4°C: ionized calcium binding adaptor molecule 1 (Iba1) (1:500, 019-19741; Wako Chemicals, Richmond, VA), CD68 (1:500, sc-135872, Santa Cruz Biotechnology), NeuN (1:500, MAB377, Millipore, Billerica, MA, USA), SREBP1 (1:100, NB100-74542, Novus Biologicals), pNFkB1 (1:100; PA5-37658, Invitrogen), and c-Cas3 (1:50; 9661, Cell Signaling Technology).

Techniques: Comparison, Immunofluorescence

Journal: Scientific Reports

Article Title: Elimination of microglia improves cognitive function following cranial irradiation

doi: 10.1038/srep31545

Figure Lengend Snippet: ( A ) Immunofluorescence staining show that PLX5622 treatment for 6 weeks reduces ~70% of CD68 + activated microglia from the control (0 Gy + PLX5622) and irradiated (9 Gy + PLX5622) brains. (a1,a2) Representative high-resolution (60×) z stacks show ramified microglial morphology in the irradiated hippocampal dentate hilus (DH) and granule cell layer (GCL) compared to 0 Gy mice that received control chow. ( B ) Quantification (Autoquant and Imaris) of CD68 + activated microglia indicated an 80–90% reduction in the control and irradiated brains receiving PLX5622 (0 Gy + PLX5622 and 9 Gy + PLX5622) at 2 week and 6 week time points. ( C ) Analysis of pro-inflammatory markers from whole brains derived from irradiated mice (0 and 9 Gy) treated with PLX5622 for 1 week at 4 week post-irradiation show radiation-induced elevation in gene expression that was reduced significantly by PLX5622 treatment. Data are presented as mean ± SEM ( N = 4 mice/group). P values are derived from ANOVA and Bonferroni’s multiple comparisons test. * P < 0.01; ** P < 0.001 compared with 0 Gy group and + P < 0.01; ++ P < 0.01compared with 9 Gy group. Scale bars: 200 μm ( A ) and 20 μm (a1,a2).

Article Snippet: For the immunofluorescence analysis of microglia, the following primary and secondary antibodies were used: rabbit anti-IBA-1 (1:500, Wako), rat anti-mouse CD68 (1:500, AbD Serotec), donkey anti-rabbit or anti-mouse conjugated with Alexa Fluor 488 or 594 (Life Technologies/Invitrogen) and DAPI nuclear counterstain (Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Control, Irradiation, Derivative Assay, Gene Expression

Journal: Scientific Reports

Article Title: Elimination of microglia improves cognitive function following cranial irradiation

doi: 10.1038/srep31545

Figure Lengend Snippet: ( A,B ) Immunofluorescence staining and laser scanning confocal microscopy for the IBA-1 + and CD68 + cells show that cranial irradiation lead to significant elevation in microglial number (40% and 25% respectively) in the pre-limbic (PrL) and infra-limbic (IL) cortices of the medial pre-frontal cortex (mPFC). (a1,b1) Representative high-resolution (60×) z stacks showed characteristic activated microglial morphology (IBA-1, a1 and CD68, b1) in the irradiated PrL compared to 0 Gy group. ( C,D ) Treatment with PLX5622 for 6 weeks eliminates 80–90% of IBA-1 + and CD68 + microglia from the control (0 Gy + PLX5622) and irradiated (9 Gy + PLX5622) mPFC. Data are presented as mean ± SEM ( N = 4 mice/group). P values are derived from ANOVA and Bonferroni’s multiple comparisons test. * P < 0.05; ** P < 0.001 compared with 0 Gy group and + P < 0.05; ++ P < 0.01compared with 9 Gy group. Scale bars: 200 μm ( A ), 100 μm ( B ) and 50 μm (a1,b1).

Article Snippet: For the immunofluorescence analysis of microglia, the following primary and secondary antibodies were used: rabbit anti-IBA-1 (1:500, Wako), rat anti-mouse CD68 (1:500, AbD Serotec), donkey anti-rabbit or anti-mouse conjugated with Alexa Fluor 488 or 594 (Life Technologies/Invitrogen) and DAPI nuclear counterstain (Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Irradiation, Control, Derivative Assay

Journal: Research Square

Article Title: Elovanoid-N32 or RvD6-isomer decrease ACE2 and binding of S protein RBD after injury or INFγ in the eye

doi: 10.21203/rs.3.rs-55764/v1

Figure Lengend Snippet: List of primary antibodies used in this study

Article Snippet: 7 , Mouse anti-rat CD68 , Bio-Rad , MCA341GA , 1:1000 , .

Techniques: Immunofluorescence, Western Blot

Journal: Biochemistry and Biophysics Reports

Article Title: Oxymatrine blocks the NLRP3 inflammasome pathway, partly downregulating the inflammatory responses of M1 macrophages differentiated from THP-1 monocytes ☆

doi: 10.1016/j.bbrep.2023.101482

Figure Lengend Snippet: Morphological images of cells in different states and identification of M0 and M1 macrophages. (A) Morphology of THP-1 cells, M0 macrophages, and M1 macrophages. Scale bar: 200 μm. THP-1 monocytes were round, translucent, and suspended; M0 macrophages were clustered and adherent; M1 macrophages were adherent and can extend pseudopodias. (B) Immunofluorescence staining on M0 cells. Scale bar: 50 μm. Colocalization of CD68 (red) and DAPI (blue). (C) Immunofluorescence staining of M1 cells. Scale bar: 50 μm. Colocalization of CD86 (red) and DAPI (blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde (PFA) (Biosharp, Anhui, China) and incubated with diluted rabbit primary antibody against CD68 (1:300) (Boster, Wuhan, China) overnight at 4 °C.

Techniques: Immunofluorescence, Staining

Journal: International Journal of Molecular Medicine

Article Title: Effects of ketanserin on experimental colitis in mice and macrophage function

doi: 10.3892/ijmm.2016.2486

Figure Lengend Snippet: 5-Hydroxytryptamine-2A receptor (5-HT 2A R) expression is upregulated in the colons of patients with inflammatory bowel disease (IBD) and in mice with dextran sodium sulfate (DSS)-induced colitis, and is specifically enhanced in macrophages. (A) Relative mRNA expression of 5-HT 2A R in healthy subjects and patients wtih IBD estimated by RT-qPCR. (B) Representative images of immunofluorescence staining of colonic mucosa samples of healthy subjects and in patients with IBD showing 5-HT 2A R + cells (in green) and CD68 + cells (in red). Co-localization of 5-HT 2A R with the macrophage marker, CD68 + , is shown in the merged images. 5-HT 2A R expression in mice with DSS-induced colitis estimated by (C) RT-qPCR and (D) western blot analysis. Representative images of immunofluorescence staining of 5-HT 2A R + cells (in green) and CD68 + cells (in red) in the colonic mucosa samples of mice treated with or without DSS. (E) Co-localization of 5-HT 2A R with CD68 is shown in the merged images.

Article Snippet: Mouse anti-human CD68 antibody (MCA5709; mouse anti-human CD68, monoclonal antibody; AbD Serotec, Kidlington, UK; 1:500) (overnight at 4°C) was subsequently used to detect the macrophages.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Marker, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Increased Atherosclerotic Lesions in LDL Receptor Deficient Mice With Hematopoietic Nuclear Receptor Rev‐erbα Knock‐ Down

doi: 10.1161/JAHA.113.000235

Figure Lengend Snippet: Rev‐erbα protein is present in the macrophages of human carotid artery atherosclerotic lesions. Human carotid artery sections were subjected to immunohistochemistry using mouse monoclonal anti‐human Rev‐erbα. A , Rev‐erbα immunoreactivity in a representative plaque (40×) (left panels), and a high‐magnification view of the same image (400×) (right panels) illustrating the cellular nature of the staining. B , Immunofluoresence staining identifying the Rev‐erbα positive cells as macrophages. A representative section of a human carotid plaque stained by using primary antibodies against Rev‐erbα (green) and the macrophage‐specific marker CD68 (red). Colocalization of the two markers is shown in yellow (left panels), and a higher magnification view of the same immunofluorescence image (right panels).

Article Snippet: After copious washing in PBS, the sections were incubated with fluorescently labeled secondary antibodies diluted in 1% (w/v) BSA in PBS for 30 minutes at 37°C (Cy3 conjugated goat antirabbit IgG [Proteintech Group Inc] for detection of CD68 antibodies; Alexa Fluor 488 conjugated goat anti‐mouse IgG [Invitrogen] for detection of Rev‐erbα antibodies).

Techniques: Immunohistochemistry, Staining, Marker, Immunofluorescence

Journal: Mucosal immunology

Article Title: A CCL24-dependent Pathway Augments Eosinophilic Airway Inflammation in House Dust Mite-challenged Cd163 −/− Mice

doi: 10.1038/mi.2015.94

Figure Lengend Snippet: (A) Confocal immunofluorescence microscopy images of BALF cells from an asthmatic subject showing CD163 expression by CD68 + AMΦs. The scale bar indicates 5 μm. (B) Gating strategy for identification of human CD163 + alveolar macrophages in BALF. Cellular debris was excluded using a forward light scatter/side scatter plot and doublets were excluded using width parameter on FSC and SSC properties. CD45 + cells, that co-expressed CD14 and CD68, were identified as alveolar macrophages using side scatter and CD45 bivariate plots from which lymphocytes had been excluded. A microscopic image of sorted CD45 + /CD14 + /CD68 + /CD163 + cells shows a cellular population possessing typical cellular characteristics of alveolar macrophages. (C) MFI of cell surface CD163 expression by CD45 + /CD14 + /CD68 + AMΦs in BALF from normal individuals and asthmatic subjects (n = 7, P < 0.008, paired t test). (D) A representative histogram overlay comparing cell surface CD163 expression by AMΦs from a normal individual and an asthmatic subject.

Article Snippet: Human BALF cell cytospin slides were fixed in 4% paraformaldehyde, incubated in blocking buffer (Aurion Blocking Solution, Electron Microscopy Sciences, Hatfield, PA) and reacted overnight with mouse anti-human CD68 (1:50 dilution, R & D Systems, Minneapolis, MN) and rabbit anti-human CD163 antibodies (1:50 dilution, Enzo Life Sciences, Inc., NY) diluted in 0.1% Aurion BSA-c (Electron Microscopy Sciences).

Techniques: Immunofluorescence, Microscopy, Expressing

Journal: Journal of Biological Chemistry

Article Title: Apolipoprotein A-I Modulates Regulatory T Cells in Autoimmune LDLr−/−, ApoA-I−/− Mice

doi: 10.1074/jbc.m110.134130

Figure Lengend Snippet: FIGURE 7. ApoA-I treatment reduces immune cell infiltrate into the skin. Panels A–D show H&E staining of skin sections from each of the four study groups; panels E–H show immunofluorescence staining for CD68 (macrophages);panelsI–LshowimmunofluorescencestainingforCD11c(dendriticcells).PanelsA,E,andIare skin sections from 12-week diet-fed SKO mice; panels B, F, and J are skin sections from 6-week diet-fed DKO mice; panels C, G, and K are skin sections from 12-week diet-fed DKO mice; panels D, H, and L are skin sections from 12-week diet-fed DKO mice treated for 6 weeks with apoA-I. Sections shown are representative of at least four sections per animal with at least 4–6 different animals per genotype and treatment group.

Article Snippet: Next, sections were incubated in their respective secondary antibody goat anti-rat IgG CD68 Cy3 (Rockland) at 1:300 or biotin-conjugated CD3e, CD11c mouse anti-hamster IgG (Pharmingen) for 45 min at room temperature.

Techniques: Staining, Immunofluorescence