Journal: EBioMedicine

Article Title: Rapid detection of bacterial infection using a novel single-tube, four-colour flow cytometric method: Comparison with PCT and CRP

doi: 10.1016/j.ebiom.2021.103724

Figure Lengend Snippet: A schematic diagram on how to calculate the FCBI-index value of the patient with microbiologically confirmed Streptococcus pneumoniae pneumonia using the novel FCBI-index method. (a) The incubation of the whole blood sample with the receptor-specific antibodies, red blood cell lysis, and flow cytometry run were carried out one after another in the same test tube. (b) During the flow cytometry run, the leukocyte population within gate R1 was first gated out from the side scatter (SSC)/MHCI bivariate histogram, after which gates R2 (red) and R3 (blue) were set around neutrophil and lymphocyte populations, respectively, in the SSC/CD64 bivariate histogram containing leukocytes inside gate R1. The stop count of 1000 monocytes inside the monocyte gate (black/R4) was used, leading to the collection of a total of 5000–20000 leukocytes. (c) For the purpose of data analysis, three additional bivariate histograms, namely SSC/CD35, SSC/CD64, and SSC/CD329 were made, showing mean fluorescence intensities (MFI, which correlate with the number of receptors on the cell surface) of gated neutrophil (red) and lymphocyte (blue) populations. In the SSC/CD35 histogram, B-lymphocytes expressing CD35 were left outside the lymphocyte gate. (d) The actual FCBI-index value was calculated by substituting the obtained receptor-specific MFI data from neutrophil and monocyte populations into the bacterial infection algorithm. A more detailed step-by-step description of the development of the FCBI-index method is presented in the method section and Supplementary Figs. 1–5. 1.54 × gCMF-HBSS: 1.54-fold calcium- and magnesium-free HBSS -buffer supplemented with 1.54 mg/mL gelatin.

Article Snippet: Fluorescence-conjugated receptor-specific monoclonal antibodies (test kit): Phycoerythrin (PE)-conjugated anti-human CD35 (Clone E11) was purchased from Ancell (ANC-184-050), PerCP-Vio700-conjugated anti-human CD64 (Clone 10.1.1) was purchased from Miltenyi Biotec (130-101-422), fluorescein isothiocyanate (FITC)-conjugated anti-human CD329 (Clone 191240) was purchased from R&D systems (FAB1139F) and AF647-conjugated anti-human MHC class I molecule (Clone W6/32) was purchased from R&D systems (FAB7098R). (Supplementary Table 5).

Techniques: Incubation, Red Blood Cell Lysis, Flow Cytometry, Fluorescence, Expressing, Infection

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: Activated FcγRI is retained in intracellular compartments described by IRAP (A–C) DC2.4 cells expressing FcγRI-GFP (green) were incubated with anti-FcγRI (clone 10.1) and cross-linked with anti-mouse IgG (blue) at 4°C. After removal of excess antibodies, the cells were shifted for the indicated time points at 37°C, fixed and stained for EEA1 (red) (A), IRAP (red) (B) or LAMP1 (red) (C). The pictures show representative images from three independent experiments and the graphs show colocalization between FcγRI and the endocytic markers. Each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Expressing, Incubation, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: Activated FcγRI recruits signaling partners at the level of IRAP endosomes (A–C) DC2.4 cells expressing FcγRI-GFP (green) were incubated with human IgG and anti-human IgG at 4°C. After removal of excess antibodies, cells were shifted for the indicated time points at 37°C, fixed and stained for: pSrc (red) and IRAP (gray) (A), LAT (red) and IRAP (gray) (B), or PLCγ (red) and IRAP (gray) (C). The pictures show representative images from three independent experiments and the graphs show colocalization between the indicated signalosome component and IRAP. Each dot represents a cell. Scale bars = 5 μm. (D) The graph shows the colocalization between FcγRI and IRAP that was quantified in the conditions (A), (B) and (C). (E) The interaction of FcγRI with Syk was measured by FLIM experiments and the graph shows the GFP lifetime for cells expressing only GFP, both GFP and mCherry as soluble proteins and the cells expressing both FcγRI-GFP and Syk-mCherry in steady state and after the receptor crosslinking for indicated time points. In all the graphs, each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Expressing, Incubation, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: IRAP deletion affects the endosomal recruitment of active Syk to FcγRI (A and B) Wild-type (WT) or IRAP-deficient (KO) BM-DCs were incubated with anti-mouse FcγRI (clone AT152-9), followed by crosslinking with non-labeled anti-rat IgG at 4°C. After removal of excess antibodies, the cells were shifted for the indicated time points at 37°C, fixed and stained for endogenous FcγRI (green) and γ-chain (red) in (A) and for γ-chain (green) and Syk phosphorylated on Tyrosine 352 (pSyk) (red) in (B). The pictures show representative images from three independent experiments and the graphs show colocalization between FcγRI and the γ-chain (A) and colocalization between the γ-chain and phosphorylated, active Syk (B). Each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Incubation, Labeling, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet:

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Produced, Western Blot, Recombinant, Lysis, Isolation, Enzyme-linked Immunosorbent Assay, Cloning, Software