Journal: Molecular and Cellular Biochemistry

Article Title: A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

doi: 10.1007/s11010-022-04557-3

Figure Lengend Snippet: 2G4 and 4C9 antibodies bind to c-Kit and inhibit c-Kit activation in LAD2 cells. A LAD2 cells were incubated with 2G4, 4C9, or normal human IgG1 at the indicated concentrations for 1 h. After washing, FITC-conjugated secondary antibody was added for 1 h. The fluorescence was detected by flow cytometry. B , C SCF-starved LAD2 cells were incubated with 2G4 antibody ( B ) or 4C9 antibody ( C ) at the indicated concentration for 1 h. Thereafter, the cells were stimulated by 100 ng/mL of SCF for an additional 10 min. Phosphorylation of c-Kit, Akt, and Erk1/2 was analyzed by Western blotting. α-Tubulin was used as a loading control

Article Snippet: The cells (2 × 10 5 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4 °C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 μg/mL, Invitrogen, CA, USA) at 4 °C for 1 h. After washing, the fluorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).

Techniques: Activation Assay, Incubation, Fluorescence, Flow Cytometry, Concentration Assay, Western Blot, Control

Journal: Molecular and Cellular Biochemistry

Article Title: A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

doi: 10.1007/s11010-022-04557-3

Figure Lengend Snippet: 2G4 antibody inhibits cell proliferation and migration in LAD2 cells. A , B LAD2 cells were incubated with 2G4, 4C9, or normal human IgG1 at the indicated concentrations in culture medium with SCF ( A ) or without SCF ( B ) for 7 days. Thereafter, cells were stained with 10 μM Hoechst 33342 and counted using a Celigo Imaging Cytometer. The black dashed line (100%) indicates normalized cell counts in the well without SCF and antibodies at 7 days. C Migration assay was carried out in a 6-transwell plate with 8 μm pores. LAD2 cells (1 × 10 6 cells) and the antibodies (1 μg/mL) were added into the upper chamber and SCF (100 ng/mL) was added into the lower chamber for 24 h. Migrated cells in the lower chamber were microscopically counted using a HPF in five different fields. All results represent the mean ± SD of three independent experiments. * vs. SCF − /Antibody − and # vs. SCF + /Antibody − . * P < 0.05, and # P < 0.05 (Student’s two-tailed t test)

Article Snippet: The cells (2 × 10 5 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4 °C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 μg/mL, Invitrogen, CA, USA) at 4 °C for 1 h. After washing, the fluorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).

Techniques: Migration, Incubation, Staining, Imaging, Cytometry, Two Tailed Test

Journal: Molecular and Cellular Biochemistry

Article Title: A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

doi: 10.1007/s11010-022-04557-3

Figure Lengend Snippet: 2G4 antibody inhibits IgE-mediated degranulation enhanced by SCF. A LAD2 cells were SCF-starved for 24 h. The cells were then incubated with 2G4, 4C9, normal human IgG1, or streptavidin for 1 h, and β-hexosaminidase release assay was performed. B , C LAD2 cells were sensitized with biotinylated human IgE ( B ) or IFN-γ ( C ) for 24 h in SCF-deficient medium. The cells were then incubated with 2G4, 4C9, normal human IgG1 or streptavidin for 1 h. Following this, β-hexosaminidase release assay was carried out. Streptavidin was used as a positive control to crosslink biotinylated-IgE. D LAD2 cells were SCF-starved and sensitized with biotinylated human IgE for 24 h. Cells were treated with antibodies (2G4, 4C9, or normal human IgG1), SCF (100 ng/mL), and streptavidin (2 ng/mL) in sequence at 30 min intervals. After 30 min of streptavidin treatment, β-hexosaminidase release assay was performed to analyze the degranulation of LAD2. All results represent the mean ± SD of three independent experiments. *, **, and *** vs Untreated, # vs. SCF − /Streptavidin − , § vs. SCF + /Streptavidin − , † vs. SCF − /Streptavidin + , and ‡ vs. SCF + /Streptavidin + . * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.05, ## P < 0.01, ### P < 0.001, § P < 0.05, §§ P < 0.01, §§§ P < 0.001, † P < 0.05, †† P < 0.01, ††† P < 0.001, ‡ P < 0.05, ‡‡ P < 0.01, and ‡‡‡ P < 0.001 (one-way ANOVA with Dunnett’s post-test)

Article Snippet: The cells (2 × 10 5 cells) were stained with 2G4, 4C9, or normal human IgG1 (Sino Biological, Beijing, China) at the indicated concentrations at 4 °C for 1 h. The cells were then rinsed thrice in PBS containing 2% BSA and stained with goat anti-human IgG secondary antibody (0.3 μg/mL, Invitrogen, CA, USA) at 4 °C for 1 h. After washing, the fluorescence signal was detected using CyFlow Cube6 (Sysmex Partec, Goerlitz, Germany), and the data analysis was performed using FCS Express 6 Flow (De Novo software, CA, USA).

Techniques: Incubation, Release Assay, Positive Control, Sequencing

Journal: bioRxiv

Article Title: Virus Entry is a Major Determinant of HCMV Latency in Monocytes

doi: 10.1101/2024.10.26.619803

Figure Lengend Snippet: (a). Flow cytometry analysis of cell surface staining of PDGFRα versus IgG control in primary and THP1 monocytes and macrophages. (b). Flow cytometry analysis of cell surface staining of PDGFRα of THP1 and THP1- PDGFRα monocytes. (c). Light microscopy of THP1 and THP1-PDGFRα monocytes. (d). Differential expression analysis of RNA-seq from THP1 and THP1-PDGFRα monocytes. Blue dots mark significant differentially expressed genes ( p < 0.05, FC > 1).

Article Snippet: FITC-conjugated Mouse IgG1 anti-human CD29 (ITGB1) (Santa cruz catalog no. MEM-101A ) and FITC-conjugated Mouse IgG1 control (Santa Cruz, catalog no. sc-2339).

Techniques: Flow Cytometry, Staining, Control, Light Microscopy, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Virus Entry is a Major Determinant of HCMV Latency in Monocytes

doi: 10.1101/2024.10.26.619803

Figure Lengend Snippet: (a). NRP2 expression in primary, THP-1 and Kasumi-3 monocytes and macrophages as measured by RNA-seq. Mono, monocytes; mac, macrophages; RPM, reads per million. (b). Flow cytometry analysis of NRP2 versus IgG control Cell surface staining in primary and THP1 monocytes and macrophages. (c). Flow cytometry analysis of THP1 macrophages, transfected with NRP2 and control siRNA two days before infection with HCMV-GFP. Analysis performed at 3 d.p.i. Quantification of the replicates is presented on the right bar graph. (d). Flow cytometry analysis of infected THP1 macrophages with NRP2 or control CRISPR knockout using HCMV-GFP virus. Analysis performed at 3 d.p.i. Quantification of the replicates is presented on the right bar graph.

Article Snippet: FITC-conjugated Mouse IgG1 anti-human CD29 (ITGB1) (Santa cruz catalog no. MEM-101A ) and FITC-conjugated Mouse IgG1 control (Santa Cruz, catalog no. sc-2339).

Techniques: Expressing, RNA Sequencing Assay, Flow Cytometry, Control, Staining, Transfection, Infection, CRISPR, Knock-Out, Virus

Journal: bioRxiv

Article Title: Virus Entry is a Major Determinant of HCMV Latency in Monocytes

doi: 10.1101/2024.10.26.619803

Figure Lengend Snippet: (a). Relative expression level of NRP2, measured by real-time PCR in THP1 macrophages treated with NRP2 siRNA compared to control siRNA at 0 h.p.i. (b) Cell surface staining with NRP2 or IgG control of THP1 macrophages treated with CRISPR knockout against NRP2 or control. Cells were analyzed by Flow cytometry.

Article Snippet: FITC-conjugated Mouse IgG1 anti-human CD29 (ITGB1) (Santa cruz catalog no. MEM-101A ) and FITC-conjugated Mouse IgG1 control (Santa Cruz, catalog no. sc-2339).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Staining, CRISPR, Knock-Out, Flow Cytometry

Journal: bioRxiv

Article Title: Virus Entry is a Major Determinant of HCMV Latency in Monocytes

doi: 10.1101/2024.10.26.619803

Figure Lengend Snippet: (a). ITGB3 and ITGB1 expression in primary, THP1 and Kasumi-3 monocytes and macrophages as measured by RNA-seq. RPM, reads per million. (b-c). Flow cytometry analysis of integrin β1 (b) and integrin β3 (c) cell surface levels in primary and THP1 monocytes and macrophages. (d). Flow cytometry analysis of control, ITGB3, ITGB1 and ITGB3 + ITGB1 knockout (KO) in THP1 macrophages infected with HCMV–GFP. Cells were analyzed at 3 d.p.i. (e). Quantification of the replicates of . (f-g). Representative Microscopy images (f) and quantification using FIJI analysis software (g) of THP1 macrophages with ITGB3 knockout (KO) versus control knockout (n=60 in the control and n=67 in the ITGB3 KO). Cells were infected with HCMV-UL32-GFP and imaged at 1.h.pi. Actin staining was used to visualize the cell’s borders and DAPI for nuclei staining. (h). Flow cytometry analysis of infected THP1 monocytes overexpressing ITGB3 and ITGAV (αvβ3), compared to mCherry control. Overexpression was induced for 24 hours using doxycycline prior to infection. Cells were analyzed at 3 d.p.i. (i). Quantification of the replicates of . (j). Expression levels of ITGB3, ITGB1 and NRP2 in CD34+ HSCs from RNA-seq of eight healthy patients . RPKM, Reads Per Kilobase per Million mapped reads.

Article Snippet: FITC-conjugated Mouse IgG1 anti-human CD29 (ITGB1) (Santa cruz catalog no. MEM-101A ) and FITC-conjugated Mouse IgG1 control (Santa Cruz, catalog no. sc-2339).

Techniques: Expressing, RNA Sequencing Assay, Flow Cytometry, Control, Knock-Out, Infection, Microscopy, Software, Staining, Over Expression

Journal: bioRxiv

Article Title: Virus Entry is a Major Determinant of HCMV Latency in Monocytes

doi: 10.1101/2024.10.26.619803

Figure Lengend Snippet: (a). Cell surface staining of THP1 macrophages with a CRISPR knockout of β3 (left), β1 (right), or both versus control knockout. Cells were analyzed by Flow cytometry. (b). real-time PCR analysis of THP1 macrophages treated with siRNA against ITGB3 or control at 0 h.p.i.. (c). Flow cytometry analysis of THP1 macrophages, transfected with ITGB3 or control siRNAs and infected with HCMV–GFP. Cells were analyzed at 3 d.p.i. (d). Quantification of the FACS replicates of . (e). Cell surface staining of THP1 overexpressing (OE) αVβ3 compared to IgG control. Cells were stained for both αV and β3 in the αVβ3 overexpressing cells. Double positive cells are gated in a box.

Article Snippet: FITC-conjugated Mouse IgG1 anti-human CD29 (ITGB1) (Santa cruz catalog no. MEM-101A ) and FITC-conjugated Mouse IgG1 control (Santa Cruz, catalog no. sc-2339).

Techniques: Staining, CRISPR, Knock-Out, Control, Flow Cytometry, Real-time Polymerase Chain Reaction, Transfection, Infection

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed in BM-DCs and LECs and supports DC transmigration. ( A ) Flow cytometry analysis of immature (−LPS) and LPS-matured (+LPS) BM-DCs (gated on live/single cells). ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression of 11 independent experiments. ( C – F ) FACS analysis of CD112 expression in ( C ) LPS-matured BM-DCs and ( E ) primary LN-LECs, derived from WT and CD112 KO mice. ( D , F ) Summary of the ∆MFI values of CD112 expression of 4–6 independent experiments. Data points of the same experiment in ( B , D , F ) are connected by a line, and the mean ΔMFI values are indicated by horizontal lines. ( G ) Set up of the transmigration experiments to investigate the transmigration of BM-DCs (WT or KO) across an LEC monolayer (WT or KO). ( H ) Impact of ICAM-1 blockade on transmigration of WT BM-DCs. ( I,J ) Impact of loss of CD112 in either ( I ) LECs or ( J ) BM-DCs on transmigration. ( K ) Impact of simultaneous loss of CD112 in LECs and BM-DCs on transmigration. For each condition in ( H – K ), one representative experiment with n = 3 technical replicates is shown on the left, and a summary of the averages of 4 independent experiments (biological replicates, each experiment in a different color) is shown on the right. Data points of the same experiment are connected by a line. ( L ) Adhesion assay of WT and KO BM-DCs to WT or KO lymphatic endothelium. The pool of two independent experiments with three replicates per condition is shown (each dot represents a sample). # BM-DCs: number of BM-DCs. Data in all graphs show mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: Transmigration Assay, Flow Cytometry, Staining, Expressing, Derivative Assay, Cell Adhesion Assay

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 expression is high in LECs but low in DCs present in murine skin. ( A , B ) FACS analysis was performed to detect CD112 expression in dermal LECs and BECs. ( A ) Depiction of the gating strategy in one representative experiment. ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression observed in 5 independent experiments. ( C – G ) Impact of TPA-induced skin inflammation on the expression of CD112 in LECs. ( C ) Schematic depiction of the experiment: Inflammation was induced in the murine ear skin by topical application of TPA and the ear skin and draining auricular LNs analyzed 24 h later. ( D – G ) FACS analyses were performed to quantify CD112 expression levels in LECs present in control or inflamed tissues. ( D , E ) Analysis of murine ear skin and ( F , G ) auricular LN single-cell suspensions. ( E , G ) The summary of ∆ MFI values was recorded in 5–6 different experiments performed in one control (CTL) and one TPA-inflamed (TPA) ear skin. ( H , I ) FACS gating and quantification of CD112 expression in DCs present in CTL and TPA-inflamed ear skin. ( H ) Gating strategy and ( I ) summary of ∆MFI values recorded in 3 different experiments. ( J – P ) Crawl-out experiments. ( J ) Schematic depiction of the experiment performed to evaluate CD112 expression in ( K – M ) DCs that had emigrated from murine ear skin into the culture medium or in ( N – P ) DCs that had remained in the cultured ear skin at the end of the experiment. Representative ( K , N ) FACS dot plots (gating on single/live cells), identifying DCs as MHCII + CD11c + cells. ( L , O ) Representative histogram plots showing CD112 expression in WT and KO DCs as well as the corresponding fluorescence minus one (FMO) control. ( M , P ) Summary of ∆MFI values (defined as specific staining—FMO) recorded in 4 different experiments performed with one WT and one KO mouse each. Data points in ( B , E , G , I , M , P ) of the same experiment are connected by a line.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: Expressing, Staining, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Loss of CD112 does not impact the in vivo migration of adoptively transferred or endogenous DCs to dLNs. ( A – D ) Adoptive transfer experiment. ( A ) Scheme of the experiment. ( B ) Gating strategy to identify fluorescently labeled adoptively transferred BM-DCs in popliteal LNs. ( C ) The ratio of KO–WT DCs recovered from popliteal LNs draining control (CTL) or CHS-inflamed (CHS) footpads of WT or KO mice. ( D – J ) FITC painting experiment. ( D ) Scheme of the experiment. ( E ) ΔEar thickness, defined as the difference between the ear thickness measured at the start and at the end of the experiment. ( F ) Cellularity and ( G ) weight of the ear-draining auricular LN at the end of the experiment. ( H ) Gating strategy to identify and quantify the number (#) of ( I ) all CD11c + MHCII hi migratory DCs (mDCs) and ( J ) FITC + mDCs. Summaries of three ( A – D ) and two ( D – J ) independent experiments, each with 2–7 mice per condition, are shown. Each dot represents one mouse. Mann–Whitney t -test was used. Red bars in all graphs show the mean. ns: not significant.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: In Vivo, Migration, Adoptive Transfer Assay, Labeling, Control, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: In the case of human DC transmigration, LECs were treated with 20 μg/mL mouse anti-human CD54 (clone: BBIG-II, R&D Systems), 20 μg/mL mouse anti-human CD112 (R2.525, Santa Cruz Biotechnology, Dallas, TX, USA) or 20 μg/mL normal mouse IgG1 (Santa Cruz) isotype control.

Techniques: Expressing, Derivative Assay, Blocking Assay, Control, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Phthiocerol Dimycocerosates From Mycobacterium tuberculosis Increase the Membrane Activity of Bacterial Effectors and Host Receptors

doi: 10.3389/fcimb.2020.00420

Figure Lengend Snippet: DIM and PMA trigger the entry of the DIM-deficient H37Rv mutant and of zymosan into macrophages through a CR3-dependent process. (A) Macrophages were either left untreated (gray square) or treated at 37°C for 1 h with 70 μM DIM (red square) or the corresponding volume of chloroform:methanol (Ø vehicle control, gray square) or for 15 min with 50 nM PMA (green square) or the corresponding volume of DMSO (Ø vehicle control, gray square). Cells were then incubated for a further 30 min with either the non-relevant IgG1 or 10 μg/mL anti-CR3 blocking antibody 2LPM19c and put in contact for 1 h with zymosan at MOI 30:1. (B) Macrophages were successively incubated with 50 nM PMA (green symbol) or the corresponding volume of DMSO (Ø vehicle control, gray symbol) and with IgG1 or 10 μg/mL 2LPM19c and then exposed to GFP-expressing H37Rv (circle) or H37Rv△ ppsE (triangle) at MOI 10:1 for 1 h. (A,B) At the end of infection, cells were rinsed, fixed and processed for the quantification of infected macrophages using a Leica 43 DM-RB epifluorescence microscope. For each set of conditions, the experiments were performed in duplicate, and at least 100 cells were counted per slide. The percentage of cells having ingested at least one bacterium, or one particle, was determined. The values are mean ± SEM of 3–7 separate experiments. The significance of difference between control and treatment was evaluated using one-way ANOVA (A) or repeated measure ANOVA (B) followed by Bonferroni's multiple comparison test; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The 2LPM19c mouse antibody directed against the CD11b subunit of human CR3 (IgG1, dilution 1:20) and normal mouse IgG1 were purchased from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Control, Incubation, Blocking Assay, Expressing, Infection, Microscopy, Comparison

Journal: Frontiers in Immunology

Article Title: Interleukin-1/-33 Signaling Pathways as Therapeutic Targets for Endometriosis

doi: 10.3389/fimmu.2019.02021

Figure Lengend Snippet: Exogenous IL-33 exacerbates endometriosis. (A) Experimental workflow. Wild type BALB/c mice were ovariectomized (OVX) and administered estrogen subcutaneously (s.c.) for 2 weeks before transplantation of uterine fragments. After transplantation, mice received PBS or recombinant human IL-33 intraperitoneally (i.p.) at day 1, 3, 5, 8, 10, and 12. For the neutralization experiment, IL-33-treated mice were intravenously (i.v.) injected with control IgG (Cont) or anti-hIL-33 Ab (αhIL-33) at day 0 and 7. (B) Representative endometriosis lesions from each mouse. (C) Total volume of the lesions ( n = 12 in each group). Pooled data from two independent experiments are shown (mean ± SD). (D) Immunohistological staining of Ki-67; Brown. L, lumen, Scale bar: 50 μm. (E) The proportion of Ki-67 positive epithelial cells lining the lumen of the cyst wall ( n = 6, mean ± SD). Statistical analyses were performed using a one-way ANOVA with Tukey's post-hoc tests (C,E) .

Article Snippet: Normal human IgG1 (Eureka therapeutics, CA) was used as a control.

Techniques: Transplantation Assay, Recombinant, Neutralization, Injection, Control, Staining