Journal: Cell metabolism

Article Title: Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

doi: 10.1016/j.cmet.2022.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD86 Antibody, anti-mouse, FITC, Miltenyi Biotec , Miltenyi Biotec , Cat# 130–123-672; RRID:AB_2889633.

Techniques: Immunofluorescence, Recombinant, Cytometry, Purification, shRNA, Cell Culture, Lysis, XF Assay, Staining, Isolation, Transfection, Live Cell Imaging, Mouse Assay, Software, Expressing

Journal: Retrovirology

Article Title: The efficiency of Vpx-mediated SAMHD1 antagonism does not correlate with the potency of viral control in HIV-2-infected individuals

doi: 10.1186/1742-4690-10-27

Figure Lengend Snippet: Effect of HIV-1 and HIV-2 on DC activation. ( A ) CD86 surface expression in DCs after infection with HIV-1 and HIV-2 IRES-eGFP constructs alone or in combination with VSV-G-pseudotyped SIVmac239 particles. The levels of CD86 expression by virally infected (eGFP+) cells were measured at 3 days post-infection. Panels A and B show mean values (±SEM) derived from four experiments. HIV-1 alone did not infect DCs at detectable levels. ( B ) Levels of IFN-γ in the supernatant of the infected DC cultures.

Article Snippet: DC activation was measured using the APC conjugated monoclonal antibody against CD86 (5397868, Invitrogen) by flow cytometry.

Techniques: Activation Assay, Expressing, Infection, Construct, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Cyanidin-3- O -Glucoside Regulates the M1/M2 Polarization of Microglia via PPARγ and Aβ42 Phagocytosis Through TREM2 in an Alzheimer’s Disease Model

doi: 10.1007/s12035-022-02873-9

Figure Lengend Snippet: The sequences of primers used to amplify target human and mouse genes

Article Snippet: Specific antibodies against CD86 (sc-19617), CD206 (sc-58986), CD163 (sc-20066), and TREM2 (sc-373828) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques:

Journal: Molecular Neurobiology

Article Title: Cyanidin-3- O -Glucoside Regulates the M1/M2 Polarization of Microglia via PPARγ and Aβ42 Phagocytosis Through TREM2 in an Alzheimer’s Disease Model

doi: 10.1007/s12035-022-02873-9

Figure Lengend Snippet: C3G regulates microglial polarization in Aβ42-induced HMC3 cells. Cells were treated with 1 μM Aβ42 and co-treated with 1 μM Aβ42 and 50 μM C3G for 24 h. a – b Relative cell surface protein expression of the M1-specific markers CD86 and CD80 and c – d M2-specific markers CD206 and CD163 was determined using flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: Specific antibodies against CD86 (sc-19617), CD206 (sc-58986), CD163 (sc-20066), and TREM2 (sc-373828) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Flow Cytometry

Journal: Molecular Neurobiology

Article Title: Cyanidin-3- O -Glucoside Regulates the M1/M2 Polarization of Microglia via PPARγ and Aβ42 Phagocytosis Through TREM2 in an Alzheimer’s Disease Model

doi: 10.1007/s12035-022-02873-9

Figure Lengend Snippet: C3G regulates microglial M1/M2 polarization via PPARγ. a HMC3 cells were treated with 1 μM Aβ42 or co-treated with 1 μM Aβ42 and 50 μM C3G. The cell surface protein expression of PPARγ was observed using flow cytometry. b – c HMC3 cells were pretreated with 10 μM GW9662 for 30 min, followed by co-treatment with 1 μM Aβ42 and 50 μM C3G for 24 h. Expression of the M1-specific marker CD86 and M2-specific marker CD206 was determined using flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: Specific antibodies against CD86 (sc-19617), CD206 (sc-58986), CD163 (sc-20066), and TREM2 (sc-373828) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Flow Cytometry, Marker

Journal: Molecular Neurobiology

Article Title: Cyanidin-3- O -Glucoside Regulates the M1/M2 Polarization of Microglia via PPARγ and Aβ42 Phagocytosis Through TREM2 in an Alzheimer’s Disease Model

doi: 10.1007/s12035-022-02873-9

Figure Lengend Snippet: C3G attenuates neuroinflammation in the cortex of APPswe/PS1ΔE9 model mice. Mice were divided into three groups: non-transgenic mice (VC), AD mice administered PBS (APPswe), and AD mice administered 30 mg/kg/day C3G (APPswe + C3G). RNA was extracted from the cortex region of mouse brains and analyzed for mRNA expression of a – c inflammatory cytokines (IL-1β, TNF-α, and IL-6), d M1-specific marker (CD86), e – f M2-specific markers (Arg1 and CD206), and g-h PPARγ and TREM2 using RT-PCR. * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: Specific antibodies against CD86 (sc-19617), CD206 (sc-58986), CD163 (sc-20066), and TREM2 (sc-373828) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transgenic Assay, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of leukocyte biology

Article Title: Adipose tissue of human omentum is a major source of dendritic cells, which lose MHC Class II and stimulatory function in Crohn's disease.

doi: 10.1189/jlb.0905501

Figure Lengend Snippet: Fig. 5. Morphology of omental DC. (a) A typical DC from a control omentum is shown. (b) An example of immunogold staining of the veils of a DC. The original bars indicate 1 m. In controls, gold labeling was always 5 gold grains per cell; 10 grains was considered positive. (c) The types of myeloid cells identified in the omental cells from two control patients; the proportion positive for HLA-DR staining is shown in the solid portion of the histograms, and those negative are hatched. M , Macrophage. (d) The gold grains per cell for the surface DR-labeling in the two experiments performed are shown. No evidence of DR-labeling was seen in either of the two Crohn’s patients studied. (e) A light microscopy picture of a cluster of putative DC immunostained with CD11c on a frozen section of omentum from a Crohn’s patient showing the dark, positive-peroxidase staining, which was absent from control slides. Cell nuclei, which were stained with hematoxylin, were distinguishable, showing that the veils were associated with mononuclear cells in the section. (f) Control for e using serum instead of specific antibody and with only hematoxylin staining visible. (g) Frozen section of control omentum, showing two cells specifically labeled for CD83 (TRITC-labeled). (h) The same section as g with label specific for CD86 (FITC-labeled).

Article Snippet: To block nonspecific binding sites, sections were incubated with normal mouse serum for 1 h and then incubated with FITC-conjugated primary antibody to CD86, CD80, or CD40 (Serotec) for 4 h, washed in PBS, and mounted under cover-slips using 4 ,6-diamidino-2-phenylindole-conjugated mounting medium (Vector Laboratories).

Techniques: Control, Staining, Labeling, Light Microscopy

Journal: Molecules (Basel, Switzerland)

Article Title: Inosine Prevents Colorectal Cancer Progression by Inducing M1 Phenotypic Polarization of Macrophages.

doi: 10.3390/molecules30010123

Figure Lengend Snippet: Figure 1. Inosine promoted macrophage polarization toward the M1 phenotype. (A) RAW264.7 cells were treated with inosine (0–40 mM) for 24 h; then, an MTT assay was performed to observe cell viability. RAW264.7 cells were administrated with inosine (1.25, 2.5, and 5 mM) in the absence or presence of LPS+IFN-γ or IL-4. (B,C) The proportions of CD86 and CD206-positive cells were determined by a flow cytometer. (D,E) The expression levels of CD86 and CD206 mRNA were detected by RT-qPCR. (F–H) Levels of CD86 and iNOS were measured by WB. Compared to the control group: * p < 0.05, ** p < 0.01, *** p < 0.001; Compared with the LPS+IFN-γ induced M1 group: # p < 0.05, ## p < 0.01, ### p < 0.001; Compared with the IL-4 induced M2 group: ∆∆∆p < 0.001.

Article Snippet: Primary antibodies against CD86 (1:1000, 26903-1-AP, Proteintech, Wuhan, China), iNOS (1:300, 22226-1-AP, Proteintech, Wuhan, China), β-actin (1:2000, GB15003-100, Servicebio, Wuhan, China) and secondary antibody against HRP Goat Anti-Rabbit IgG (1:5000, AS014, ABclonal, Wuhan, China) were used, respectively.

Techniques: MTT Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Control

Journal: Molecules (Basel, Switzerland)

Article Title: Inosine Prevents Colorectal Cancer Progression by Inducing M1 Phenotypic Polarization of Macrophages.

doi: 10.3390/molecules30010123

Figure Lengend Snippet: Figure 4. Effects of inosine on immune factors in the CT26 tumor microenvironment. (A) Effect of inosine on Ki-67 expression in tumor tissues (Scale: 100 µm; 400× and 200×). (B) Statistics of Ki-67 protein positive expression in tumor tissues (n = 3). (C) Fluorescence co-localization fluorogram of M1- type macrophage marker F4/80 + CD86 (scale: 100 µm; 200×). (D) F4/80 + CD86 expression statistics in tumor tissues. (E) M2 type macrophage marker F4/80 + CD206 fluorescence co-localization fluorogram (scale: 100 µm; 200×). (F) F4/80 + CD206 expression statistics in tumor tissues. Note: 5-Fu are 5-Fu (12 mg/kg) groups. IS-L and IS-H are inosine low and high-dose (5 mg/kg and 50 mg/kg) groups, respectively. Yellow arrows represent positive positions. Compared with the model group: ## p < 0.01; Compared with the 5-Fu group: △p < 0.05.

Article Snippet: Primary antibodies against CD86 (1:1000, 26903-1-AP, Proteintech, Wuhan, China), iNOS (1:300, 22226-1-AP, Proteintech, Wuhan, China), β-actin (1:2000, GB15003-100, Servicebio, Wuhan, China) and secondary antibody against HRP Goat Anti-Rabbit IgG (1:5000, AS014, ABclonal, Wuhan, China) were used, respectively.

Techniques: Expressing, Fluorescence, Marker

Journal: Acta biochimica et biophysica Sinica

Article Title: Tissue extracellular vesicles suppress neonatal cardiac regeneration: a Pak2-Erk1/2-mediated macrophage paracrine signaling.

doi: 10.3724/abbs.2024193

Figure Lengend Snippet: Figure 6. P8-EVs inhibit cardiomyocytes proliferation by modulating the macrophage phenotype (A) Flow cytometry analysis of macrophages internalizing P1/P8-EVs expressing the M1 macrophage polarization marker CD86. (B) The proportion of CD86-positive macrophages (n = 3). (C) The phagocytic function of macrophages was assayed with pHrodo dye (red). Scale bar: 50 μm. (D) mRNA expression levels of CD86, CCR2, Areg and OSM (n = 3). (E) Coculture system of P1/P8-EVs, macrophages and cardiomyocytes. (F) Phalloidin (green) and DAPI (blue) staining of mac- rophages internalized Dil-labelled (red) P1/P8-EVs. Scale bar: 10 μm. (G) CTNT (red), KI67 (cyan) and DAPI (blue) staining of cardiomyocytes co- culture with differently treated macrophages. There were 36 Ki67-positive cells in 137 cardiomyocytes in the control group; 36 Ki67-positive cells in 129 cardiomyocytes in the P1 group; 17 Ki67-positive cells in 119 cardiomyocytes in the P8 group; and 31 Ki67-positive cells in 120 cardiomyocytes in the P8+Pak2 inhibitor group. Scale bar: 20 μm. (H) Immunofluorescence staining of CTNT (red), BrdU (cyan) and DAPI (blue) in cardiomyocytes co-culture with differently treated macrophages. 46 Ki67-positive cells out of 191 cardiomyocytes in the control group; 35 Ki67-positive cells out of 147 cardiomyocytes in the P1 group; 13 Ki67-positive cells out of 100 cardiomyocytes in the P8 group; 21 Ki67-positive cells out of 95 cardio- myocytes in the P8 + Pak2 inhibitor group. Scale bar: 20 μm. (G′-H′) Statistical analysis of the above data (n = 3).

Article Snippet: The cells were collected, rinsed with PBS, processed with an antibody against CD86 (1:200 dilution; Proteintech, Wuhan, China), and centrifuged.

Techniques: Flow Cytometry, Expressing, Marker, Staining, Co-Culture Assay, Control, Immunofluorescence

Journal: ACS Applied Materials & Interfaces

Article Title: Acceleration of Oral Wound Healing under Diabetes Mellitus Conditions Using Bioadhesive Hydrogel

doi: 10.1021/acsami.2c17424

Figure Lengend Snippet: Fe-TA@P(AM-AA) improves the inflammatory microenvironment of diabetic oral wounds in vivo. (A) Scheme for the creation and covering of a diabetic rat palatal mucosal defect model to assess the wound-healing potential of gels. (B) H&E staining showing the successful creation of mucosal defects of the same size in the epithelium and lamina propria (labeled with dashed arrows) on both sides of the palatal mucosa. (C) CD16-stained neutrophils in the defect regions, with higher-magnification images of the regions marked by red boxes shown on the right. The percentage of CD16-positive cells was measured. (D) CD206-stained M2 macrophages in the defect regions, with higher-magnification images of the regions marked by red boxes shown on the right. The percentage of CD206-positive cells was measured. (E) CD86-stained M1 macrophages in the defect regions, with higher-magnification images of the regions marked by red boxes shown on the right. The percentage of CD86-positive cells was measured.

Article Snippet: Subsequently, they were blocked with 5% bovine serum albumin (BSA) for 1 h, followed by incubation with primary antibodies anti-CD86 (ABclonal, A1199, 1:200) and anti-CD206 (Proteintech, 60143-1-Ig, 1:200), respectively, at 4 °C overnight.

Techniques: In Vivo, Staining, Labeling

Journal: Materials Today Bio

Article Title: Antibacterial coaxial hydro-membranes accelerate diabetic wound healing by tuning surface immunomodulatory functions

doi: 10.1016/j.mtbio.2022.100395

Figure Lengend Snippet: Ag-PCL/GelMA fibrous membranes impair AGEs-induced macrophages M1 polarization and increased macrophages M2 polarization A) The images of BMDMs left untreated (Left) or treated with LPS (Center) or AGEs (Right). Scale bar, 50 ​μm. B) Fluorescence images of phalloidin-labelled BMDMs. Scale bar, 50 ​μm (first panel); 10 ​μm (second panel). C) Scheme of cell morphology change and definition of cell elongation. D) Quantification of elongation, or length of the long axis divided by length of the short axis, for control, LPS-treated, and AGEs-treated cells. n ​= ​50. E) Quantitative cell area based on fluorescence images phalloidin-labelled cells. F) Fluorescence images of BMDMs stained for iNOS (green) and CD 86 (red) and DAPI nuclear counterstain (blue) of control (Left), LPS-treated (Center) and AGEs-treated cells (Right). Scale bar, 50 ​μm. G) Representative Western blot of iNOS and CD 86 of control, LPS-treated, and AGEs-treated cells. H) The grey levels of iNOS and CD86 were analyzed and normalized to GAPDH. I) Fluorescence images of BMDM stained for CD86 (red) and DAPI nuclear counterstain (blue). And quantitative fluorescence intensity of CD86 of macrophages on different samples. Scale bars, 50 ​μm. J) Fluorescence images of BMDM stained forCD206 (green) and DAPI nuclear counterstain (blue). And quantitative fluorescence intensity of CD206 of macrophages on different samples. Scale bars, 50 ​μm. K) Fluorescence images of BMDMs stained for iNOS (red) and DAPI nuclear counterstain (blue). And quantitative fluorescence intensity of iNOS of macrophages on different samples. Scale bars, 50 ​μm. L) Fluorescence images of BMDMs stained for Arg-1(green) and DAPI nuclear counterstain (blue). And quantitative fluorescence intensity of Arg-1 of macrophages on different samples. Scale bars, 50 ​μm. M) ELISA analyses of anti-inflammatory cytokines IL-10. N) ELISA analyses of anti-inflammatory cytokines TGF-β. O) ELISA analyses of anti-inflammatory cytokines IL-6. P) ELISA analyses of anti-inflammatory cytokines TNF-α. n ​= ​3. ∗P ​< ​0.05 and ∗∗P ​< ​0.01. (For interpretation of the references to colour/colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies rabbit anti-CD86 (1:200, Abcam, USA), rabbit anti-iNOS (1:200, Abcam, USA), rabbit anti–NF–κB p65 (1:200, Cell Signaling Technology, USA) and rabbit anti-phospho–NF–κB p65 (1:200, Cell Signaling Technology, USA) were applied to samples at 4 ​°C overnight and washed off thereafter.

Techniques: Fluorescence, Staining, Western Blot, Enzyme-linked Immunosorbent Assay