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: Glia

Article Title: Activated microglia do not increase 18 kDa translocator protein ( TSPO ) expression in the multiple sclerosis brain

doi: 10.1002/glia.24052

Figure Lengend Snippet: Antibodies for immunohistochemistry

Article Snippet: CD68 , Rabbit , Atlas Antibody , HPA048982 , 1:4000.

Techniques:

Journal: PLOS One

Article Title: Photobiomodulation therapy increases neural stem cell pool in aged 3xTg-AD mice

doi: 10.1371/journal.pone.0321668

Figure Lengend Snippet: A. Representative confocal images of Aβ plaques (6E10), microglia (Iba1) and CD68 showing diffuse microglial phagocytosis away from plaques in sham compared to localized phagocytosis around plaques in PBM-treated animals. B. Quantitative analysis of total and activated microglia show no overall differences in the neuroinflammatory environment surrounding Aβ plaques. (Unpaired t-test). C. Analysis of the distribution of CD68+ microglia shows a trend of higher concentration of phagocytic cells in contact with plaques, and decreasing phagocytosis away from plaques. (Two-way ANOVA).

Article Snippet: Up to six sections representing different regions of the hippocampus were immunostained with the following primary antibodies: Sox2 (Abcam), doublecortin (DCX, Santa Cruz), calretinin (CR, SWANT), β-amyloid (6E10, Biolegend), phospho-tau (pTau, AT8, Invitrogen), βIII-tubulin (Abcam), Iba1 (Synaptic Systems), and CD68 (Rockland).

Techniques: Concentration Assay

Journal: Cancers

Article Title: Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells

doi: 10.3390/cancers13133298

Figure Lengend Snippet: Immunohistochemical characterization of immune infiltrate in MLPS tissues. Representative images of H&E and IHC staining of CD3+, CD4+, CD8+, CD68+, and CD163+ positive cells in FFPE sections from a low grade MLPS ( a ) from patient #21 and high grade. MLPS tissues ( b ) from patient #4, acquired at 200× magnification.

Article Snippet: Monocytes recovered from co-cultures with/without MLPS cells were analyzed by flow cytometry as described [ ], using PE-conjugated anti-CD68 REAfinity™ (Miltenyi Biotec #130-114-460) and APC-conjugated anti-CD163 REAfinity™ (Miltenyi Biotec, #130-112–129, Bergisch Gladbach, Germany) antibodies.

Techniques: Immunohistochemical staining, Immunohistochemistry

Journal: Cancers

Article Title: Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells

doi: 10.3390/cancers13133298

Figure Lengend Snippet: Correlation between immune cell infiltration and microvessel density in 26 low grade versus 24 high grade MLPS tissues. Box plots, showing variation in the average count of CD3+, CD4+, CD8+, FOXP3+, CD68+, and CD163+ cells as well as CD31 positive microvessels according to low or high histologic grade. Dark horizontal lines indicate the medians. Circles indicate outliers.

Article Snippet: Monocytes recovered from co-cultures with/without MLPS cells were analyzed by flow cytometry as described [ ], using PE-conjugated anti-CD68 REAfinity™ (Miltenyi Biotec #130-114-460) and APC-conjugated anti-CD163 REAfinity™ (Miltenyi Biotec, #130-112–129, Bergisch Gladbach, Germany) antibodies.

Techniques:

Journal: Cancers

Article Title: Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells

doi: 10.3390/cancers13133298

Figure Lengend Snippet: Pearson correlation between the average count of CD163+ macrophages and ( a ) CD3+ T lymphocytes, ( b ) CD4+ T helper lymphocytes, ( c ) CD8+ cytotoxic lymphocytes, ( d ) CD68+ macrophages, and ( e ) CD31+ microvessels in 50 MLPS tissue sections. Pearson correlation coefficients (r) are indicated.

Article Snippet: Monocytes recovered from co-cultures with/without MLPS cells were analyzed by flow cytometry as described [ ], using PE-conjugated anti-CD68 REAfinity™ (Miltenyi Biotec #130-114-460) and APC-conjugated anti-CD163 REAfinity™ (Miltenyi Biotec, #130-112–129, Bergisch Gladbach, Germany) antibodies.

Techniques:

Journal: Cancers

Article Title: Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells

doi: 10.3390/cancers13133298

Figure Lengend Snippet: MLPS cells trigger M2-like polarization of monocytes in non-contact co-cultures. ( a , b ) Representative images of H&E staining of FFPE sections from #37 ( a ) and #47 ( b ) MLPS tissues acquired at 200× magnification. ( c , d ) Primary MLPS cells obtained by enzymatic digestion of 37 ( c ) and #47 ( d ) tumor tissues, visualized by phase contrast microscopy (left) and fluorescent microscopy after immunostaining with anti-vimentin and anti-cytokeratin Abs (right). Nuclei were stained blue with DAPI. Original magnifications: 200× (left) and 400× (right). ( e , f ) Human monocytes were co-cultured with #37 ( e ) and #47 ( f ) primary MLPS cells in an in vitro non-contact co-culture for 72 h and then analyzed for CD68 and CD163 expression by flow cytometry. ( e , f ) Percent variation of CD68 and CD163 on monocytes collected after non-contact co-culture, compared to control monocytes. ( g , h ) After co-cultures with #37 ( g ) and #47 ( h ) primary MLPS cells, CMs from monocytes were analyzed for the content of CC2, IL-10, and IL-12 by a dot plot assay. The pixel density of each spot was measured using NIH Image J 2.0 software developed by the US NIH, USA and positive control spots were used to normalize results between the membranes. The intensity of each spot was averaged over the duplicate spots and expressed as percentage of each cytokine or chemokine spontaneously secreted by control monocytes (monocyte CM), considered as 100% (dashed line). Data represent mean ± SD from three experiments performed in duplicate with * p < 0.05, ** p < 0.005.

Article Snippet: Monocytes recovered from co-cultures with/without MLPS cells were analyzed by flow cytometry as described [ ], using PE-conjugated anti-CD68 REAfinity™ (Miltenyi Biotec #130-114-460) and APC-conjugated anti-CD163 REAfinity™ (Miltenyi Biotec, #130-112–129, Bergisch Gladbach, Germany) antibodies.

Techniques: Staining, Microscopy, Immunostaining, Cell Culture, In Vitro, Co-Culture Assay, Expressing, Flow Cytometry, Software, Positive Control

Journal: The American journal of pathology

Article Title: The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

doi: 10.1016/j.ajpath.2017.10.015

Figure Lengend Snippet: Figure 9 Transient versican G1 fragmenteserum-derived hyaluronan-associated proteinehyaluronan (VG1F-SHAP-HA) complex formation in a mouse model of moist wound healing. A: Representative images from wound tissues from the moist wound-healing model are shown. Edematous granulation tissues were observed at day 3 after wounding; however, the granulation tissues were nonedematous and thin at days 1 and 6. The thickness of the granulation tissues is as follows: day 1, 0.77 0.10 mm; day 3, 2.55 0.17 mm; and day 6, 0.82 0.04 mm. B: Tissue specimens were stained with hematoxylin and eosin. C: Isolated tissues were stained by using the indicated probes. The VG1F-SHAP-HA complex was observed on day 3 in specific lesions, but was absent at days 1 and 6. The color of letters indicating molecules represents the color of the stain. The merged images indicate the VG1F-SHAP-HA complex. D: Immunohis- tochemical analysis of wound tissue taken from day 3. The specimen was stained with the indicated probes, and the merged images show VG1F colocalization, as detected by probes for HA, DPEAAE epitope, and macrophages (CD68). The merged images show macrophage/complex colocalization. Data are expressed as means SD (A). n Z 4 independent experiments (A). Scale bars Z 50 mm (BeD). HC, heavy chain.

Article Snippet: A goat pAb against bikunin (sc-21597) was purchased from Santa Cruz Biotechnology Inc. A mouse monoclonal antibody against the macrophage marker CD68 (STJ-6572) was purchased from St. John’s Laboratory Ltd (London, UK).

Techniques: Derivative Assay, Staining, Isolation

Journal: The American journal of pathology

Article Title: The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

doi: 10.1016/j.ajpath.2017.10.015

Figure Lengend Snippet: Figure 11 The versican G1 fragment (VG1F)eserum-derived hyaluronan (HA)eassociated proteineHA complex in human pressure ulcers. A: In human pressure ulcers, edematous wounds (e1 and e2) typically become flat and stabilized (f3 and f4) after appropriate treatments. B: The VG1F-HC complex was detected from the protein extract of wound surfaces shown in A (e1 and e2). The samples are indicated at the top of the figure and correspond to those shown in A. The samples were extracted with 6 mol/L GdnHCl and treated with trypsin and V8 protease (Materials and Methods); separated using SDS-PAGE under nonreducing conditions; and blotted. Incubation with the indicated antibodies revealed the cross-linked heavy chain (HC) and VG1 species (dagger). The bands of cross-linked HC and VG1 species (dagger) disappeared during wound healing. C: Paired samples from edematous or flat wounds were analyzed using Western blotting, and the bands shown in B were quantified. The relative intensities of the bands [VG1-HCs (daggers in B)]/the total polyclonal antibody 6084epositive bands (all VG1) differ significantly (P Z 0.02) between edematous and flat wounds. D: Direct immunofluorescence of wound surface samples obtained from different sites of one individual wound. Samples obtained from the edematous wound (green dashed circle) and flat wound (blue dashed circle) were fixed and incubated with VG1 and HC1 antibodies, as well as biotin-conjugated HAebinding protein, as indicated. A representative result from triplicate experiments with different samples is shown. E: Immunohistochemical analysis of pressure ulcer tissue. The specimen was stained, as indicated. The merged images show VG1F colocalization, as detected by antibodies for DPEAAE epitope, HC1, and macrophage CD68. Data are expressed as means SD (C). n Z 10 (C, each paired sample). Scale bars: 5 mm (D); 100 mm (E).

Article Snippet: A goat pAb against bikunin (sc-21597) was purchased from Santa Cruz Biotechnology Inc. A mouse monoclonal antibody against the macrophage marker CD68 (STJ-6572) was purchased from St. John’s Laboratory Ltd (London, UK).

Techniques: Derivative Assay, SDS Page, Incubation, Western Blot, Immunohistochemical staining, Staining