Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Expression of CD86 on human islet endothelial cells facilitates T cell adhesion and migration.

doi: 10.4049/jimmunol.181.9.6109

Figure Lengend Snippet: FIGURE 1. Phenotypic characterization of primary cultures of human islet ECs. A and B, The morphology of islet ECs at confluence observed by light microscopy (phase contrast) (A) and the expression of endothelial surface phenotypic markers on cultured human islet ECs including CD105, CD31, CD146 and E-selectin (B). C and D, The expression of MHC class I (HLA-ABC), MHC class II (HLA-DR), ICAM-1, and VCAM-1 adhesion molecules on resting islet ECs (C) and the basal expression of costimulatory molecules including CD40, CD80, CD86, and ICOS-L on resting islet ECs (D). Empty histograms represent staining with isotype-matched control mAbs. Study data are representative of at least three separate flow cytometric analyses conducted on islet ECs from all three islet donors.

Article Snippet: Analysis of the immune phenotype of islet endothelial cells For the analysis of the surface expression of immune phenotypic markers, confluent EC monolayers were detached from wells with Accutase (TCS CellWorks), washed in PBS/2% FCS, and stained for 30 min at 4°C with fluorochrome-conjugated mouse anti-human CD40, CD54, CD62E, and CD86 (all from Serotec); mouse anti-human CD80, CD106, HLA-DR, and HLA-ABC (all BD Biosciences and BD PharMingen); and mouse antihuman ICOS-L (Lab Vision) or appropriate isotype control mAbs.

Techniques: Light Microscopy, Expressing, Cell Culture, Staining, Control

Journal: Journal of Neuroinflammation

Article Title: Exosomes derived from atorvastatin-modified bone marrow dendritic cells ameliorate experimental autoimmune myasthenia gravis by up-regulated levels of IDO/Treg and partly dependent on FasL/Fas pathway

doi: 10.1186/s12974-016-0475-0

Figure Lengend Snippet: Distribution of Dex after injection into the EAMG rats and effects of statin-Dex injection on endogenous DCs. The PKH26-labeled ( red ) Dex were detected in the spleen, thymus, and popliteal and inguinal lymph nodes on days 1 and 3 after injection ( a , original magnification ×100). The expression of CD80, CD86, and MHC class II on endogenous DCs on days 1 and 3 after injection were examined by FACS. The results showed that endogenous DCs from EAMG rats of statin-Dex group expressed lower level of CD80 on day 1 after injection and lower levels of CD80, CD86, and MHC class II on day 3 after injection when compared with those from control-Dex group. There were no significant differences for the levels of CD86 and MHC class II expressed on endogenous DCs from EAMG rats on day 1 after injection between statin-Dex group and control-Dex group ( b ). To further investigate the mechanism of exogenous Dex injection on endogenous DCs, spleen-derived DCs from ongoing EAMG rats were cultured with PKH26-labeled ( red ) statin-Dex and control-Dex in vitro, respectively. The percentages of PKH26-labeled endogenous DCs were examined by flow cytometry ( c ). Meanwhile, these DCs were labeled with mouse anti-rat OX62 antibody and FITC-conjugated anti-mouse IgG antibody ( green ). Then, the labeled DCs were examined by fluorescence microscopy. Both statin-Dex and control-Dex could be internalized by or fused with spleen-derived DCs in vitro ( red stands for Dex, green stands for DCs, and yellow refers to overlap of red and green) (The diameter of spleen-derived DCs from rats is about 10–20 μm) ( d , original magnification ×400). The results are expressed as mean ± SD ( n = 5 rats per group) (* p < 0.05 and *** p < 0.001)

Article Snippet: Thereafter, we labeled these spleen-derived DCs (endogenous DCs) with FITC-conjugated anti-rat CD80 (AbD Serotec, Oxford, UK), FITC-conjugated anti-rat CD86 (BioLegend), and FITC-conjugated anti-rat MHC class II (eBioscience) for 30 min at 4 °C.

Techniques: Injection, Labeling, Expressing, Control, Derivative Assay, Cell Culture, In Vitro, Flow Cytometry, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Chlorin e6-Induced Photodynamic Effect Polarizes the Macrophage Into an M1 Phenotype Through Oxidative DNA Damage and Activation of STING

doi: 10.3389/fphar.2022.837784

Figure Lengend Snippet: Ce6 PDT remodeled macrophages into the M1 phenotype in vitro . Macrophages were treated by Ce6 PDT (Ce6 was loaded at a concentration of 4 μg/ml, the loading time was 12 h, the irradiation time was 20/40/60 s, and the placement time was 8 h). (A,B) Phagocytic ability of macrophages was assayed through the fluorescence latex beats experiment and confocal microscopy. The average number of latex beats engulfed by macrophages was counted. (C–E) Expressions of GBP5 and iNOS were measured by WB. The blots were quantitatively analyzed using the ratio of mean gray. (F,G) mRNA levels of IL-1β and IL-6 were quantified through RT-PCR. (H–M) Surface CD80, CD86, and MHC-II expressions were detected by immunofluorescence staining and flow cytometry. Geometric means were used to quantify the MFI. (N,O) LLC were co-cultured with macrophages treated with Ce6, as described before. The apoptosis rate of LLC was detected by Annexin-V/PI double staining and flow cytometry. Values were means ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: For the detection of NF-κB, LC-3, and MHC-II expressions or location, cells were incubated with primary antibodies of NF-κB (10745-1-AP, Proteintech, Wuhan, China), LC-3 (12741S, CST, Boston, United States), CD80 (E-AB-F0992D, Elabscience), CD86 (E-AB-F0994D, Elabscience), and MHC-II (sc-66205, Santa Cruz Biotechnology, Santa Cruz, United States) overnight at 4°C and then incubated with the goat anti-rat IgG/Alexa Fluor 488 secondary antibody (bs-0293G-AF488, Bioss, Beijing, China) for another 60 min and washed three times before confocal microscopy (FV3000RS, Olympus, Japan) or flow cytometry (CytoFLEX, Beckman Coulter, United States).

Techniques: In Vitro, Concentration Assay, Irradiation, Fluorescence, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Double Staining

Journal: Frontiers in Pharmacology

Article Title: Chlorin e6-Induced Photodynamic Effect Polarizes the Macrophage Into an M1 Phenotype Through Oxidative DNA Damage and Activation of STING

doi: 10.3389/fphar.2022.837784

Figure Lengend Snippet: Suppression of the STING molecule attenuated the Ce6 PDT-induced M1 phenotype remodeling of macrophages. The STING molecule in macrophages was silenced by SiRNA (100 pM) for 8 h and then treated with Ce6, as described before. (A–C) The expression of cGAS and p-NF-κB in macrophages was measured by WB. The blots were quantitatively analyzed using the ratio of mean gray. (D–F) The molecules of iNOS and GBP5 were analyzed by WB. The blots were quantitatively analyzed using the ratio of mean gray. (G,H) The expression of the STING was detected by WB. The blots were quantitatively analyzed using the ratio of mean gray. (I–N) The surface expression of MHC-II, CD80, and CD86 was analyzed by immunofluorescence staining and flow cytometry. Geometric means were used to quantify the MFI. Values were means ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: For the detection of NF-κB, LC-3, and MHC-II expressions or location, cells were incubated with primary antibodies of NF-κB (10745-1-AP, Proteintech, Wuhan, China), LC-3 (12741S, CST, Boston, United States), CD80 (E-AB-F0992D, Elabscience), CD86 (E-AB-F0994D, Elabscience), and MHC-II (sc-66205, Santa Cruz Biotechnology, Santa Cruz, United States) overnight at 4°C and then incubated with the goat anti-rat IgG/Alexa Fluor 488 secondary antibody (bs-0293G-AF488, Bioss, Beijing, China) for another 60 min and washed three times before confocal microscopy (FV3000RS, Olympus, Japan) or flow cytometry (CytoFLEX, Beckman Coulter, United States).

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Science advances

Article Title: Cationic nanoparticles enhance T cell tumor infiltration and antitumor immune responses to a melanoma vaccine.

doi: 10.1126/sciadv.abk3150

Figure Lengend Snippet: Fig. 5. In vitro stimulation of BMDCs. BMDCs were incubated for 2 days with the indicated treatment (as described in Materials and Methods), and then cells and supernatants were harvested for analysis of surface marker expression and cytokine secretion, respectively. (A) Graph showing relative CD40 expression. (B) Graph showing relative CD80 expression. (C) Graph showing percent CD86hi expression. (D) Graph showing relative MHC class I expression. (E) Graph showing IL-6 secretion levels. (F) Graph showing difference in IP-10 secretion. Data are plotted as means ± SD. Probability values are determined by one-way ANOVA with Tukey posttest.

Article Snippet: BMDCs were then incubated with one or more of the following antibodies: anti-mouse CD11c fluorescein isothiocyanate (FITC) (clone N418, Invitrogen), anti-mouse CD40 APC (clone 3/23, BioLegend, San Diego, CA), anti-mouse CD80 APC (16-10A1), antimouse CD86 APC (20-0862, Tonbo Biosciences, San Diego, CA), and anti-mouse MHC class I (H-2Kb) PE (AF6-88.5.5.3, Invitrogen, Waltham, MA), for 30 min on ice in the dark (72).

Techniques: In Vitro, Incubation, Marker, Expressing

Journal: BMC immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment.

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: Fig. 3 TRPV1+ neurons affect the polarization of local macrophages in the skin. a IHC staining of the skin of TRPV1−/− and WT mice; scale bars, 500 μm; high magnification images, scale bars, 50 μm. b-d IHC analysis of the ratio of M0 (F4/80 +), M1 (CD80 +) and M2 (CD206 +) cells in TRPV1−/− and WT mice (n = 5/group); unpaired t test. e–h Flow cytometric analysis of M0 (CD11b + F4/80 +), M1 (CD11b + F4/80 + CD80 +), and M2 (CD11b + F4/80 + CD206 +) ratios in the skin of TRPV1−/− and WT mice (n = 5/group); unpaired t test. i-k Tissue expression levels of TNFα, IL-1β and IL-10 in TRPV1−/− and WT mice 1 day after skin infection; the control group was uninfected WT mice; One-way ANOVA with Tukey’s post hoc test. Data were pooled from two or three independent experiments

Article Snippet: After washing, the cells were fixed/permeabilized according to the instructions provided by the manufacturer (MultiSciences, FoxP3/Transcription Factor Staining Buffer Kit) and then incubated on ice with CD206 (Elabscience, #E-AB-F0992C) for 30 min. Flow cytometry data were collected and exported using Beckman CytoFLEX (USA).

Techniques: Immunohistochemistry, Expressing, Infection, Control

Journal: BMC immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment.

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: Fig. 5 CGRP regulates the polarization of BMDMs and the release of inflammatory factors. a-c BMDMs cultured in vitro were induced to M1 polarization with IFN-γ and M2 polarization with IL-4 The polarized macrophages were treated with CGRP or PBS and stained with DAPI (blue), CD80 (green), and CD206 (red); the ratio of CD80 + and CD206 + was analyzed under different intervention conditions. No difference in the confluency of BMDMs was observed among different groups. Scale bars, 20 μm. One-way ANOVA with Tukey’s post hoc test. d-f Expression levels of TNFα, IL-1β, and IL-10 in BMDMs after polarization in vitro under different experimental conditions; one-way ANOVA with Tukey posttests. Data were pooled from two or three independent experiments

Article Snippet: After washing, the cells were fixed/permeabilized according to the instructions provided by the manufacturer (MultiSciences, FoxP3/Transcription Factor Staining Buffer Kit) and then incubated on ice with CD206 (Elabscience, #E-AB-F0992C) for 30 min. Flow cytometry data were collected and exported using Beckman CytoFLEX (USA).

Techniques: Cell Culture, In Vitro, Staining, Expressing

Journal: Cancer Immunology, Immunotherapy

Article Title: Cryptotanshinone has curative dual anti-proliferative and immunotherapeutic effects on mouse Lewis lung carcinoma

doi: 10.1007/s00262-019-02326-8

Figure Lengend Snippet: CT induced maturation of mouse DCs. a Mouse bone marrow-derived DCs were incubated in a CO 2 incubator for 48 h with or without CT or LPS at the concentrations specified before they were immunostained for detection of surface marker (CD80, CD83, CD86, and I-A/E) expression by flow cytometry. Shown are the overlay histograms of sham (blue line) and treated (red line) DCs. b Mouse bone marrow-derived DCs were cultured in the absence (sham) or presence of various concentrations of CT for 24 or 48 h before the supernatants were harvested for the measurement of indicated cytokines. Shown is the average (mean ± SD) of three independent experiments. * p < 0.05 and ** p < 0.001

Article Snippet: Mouse DCs were stained with FITC-anti-mouse CD86 (clone GL1, TONBO Biosciences, San Diego, CA), PE-anti-mouse CD80 (clone 16-10A1, TONBO), Pacific Blue-anti-mouse CD83 (clone Michel-19, BD), and APC-anti-mouse I-A/E (clone M5/114.15.2, eBioscience).

Techniques: Derivative Assay, Incubation, Marker, Expressing, Flow Cytometry, Cell Culture

Journal: Cancer Immunology, Immunotherapy

Article Title: Cryptotanshinone has curative dual anti-proliferative and immunotherapeutic effects on mouse Lewis lung carcinoma

doi: 10.1007/s00262-019-02326-8

Figure Lengend Snippet: Comparison of CT-induced maturation of wild-type (WT), TLR4 −/− , and MyD88 −/− mouse DCs. a , WT (C57BL/6), TLR4 −/− , and MyD88 −/− mouse DCs were incubated in a CO 2 incubator for 48 h with CT (5 µg/ml) or LPS (100 ng/ml) before they were immunostained for the detection of surface marker (CD80, CD83, CD86, and I-A/E) expression by flow cytometry. Shown are the overlay histograms of sham (blue line) and treated (red line) DCs. b , Mouse bone marrow-derived DCs were cultured in the absence (sham) or presence of CT (5 µg/ml) or LPS (100 ng/ml) for 48 h and the supernatants were harvested to assay the indicated cytokines. Shown is the average (mean ± SD) of three independent experiments. * p < 0.05 and ** p < 0.001

Article Snippet: Mouse DCs were stained with FITC-anti-mouse CD86 (clone GL1, TONBO Biosciences, San Diego, CA), PE-anti-mouse CD80 (clone 16-10A1, TONBO), Pacific Blue-anti-mouse CD83 (clone Michel-19, BD), and APC-anti-mouse I-A/E (clone M5/114.15.2, eBioscience).

Techniques: Comparison, Incubation, Marker, Expressing, Flow Cytometry, Derivative Assay, Cell Culture