Journal: PLoS ONE

Article Title: Syntaxin Binding Protein 1 Is Not Required for Allergic Inflammation via IgE-Mediated Mast Cell Activation

doi: 10.1371/journal.pone.0058560

Figure Lengend Snippet: A, Wild type, STXBP1 mutant, or heterozygous mice were identified by PCR using DNA from liver cells. B , After 4–5 weeks of culture, liver-derived mast cells (LMC) were examined by toluidine blue staining to determine mast cell purity. A representative photomicrograph is shown (original magnification = ×100). C , LMCs were sensitized with anti-TNP IgE, stained with FITC-conjugated anti-IgE or anti-c-kit antibodies, and examined by flow cytometry. D, STXBP1 +/+ and STXBP1 −/− LMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA (10 ng/ml) for various times. Whole cell lysates were analyzed by Western blotting for STXBP1 and Actin. E, Sec1/Munc18 (SM) family gene expression profiles were identified by RT-PCR using mRNA from wild type mouse bone marrow-derived mast cells (BMMC), and wild type (STXBP1 +/+ ) or STXBP1 −/− LMCs, with or without TNP-BSA stimulation. All members of SM family are expressed in wild type BMMCs and LMCs. Similar results were observed in 2 independent experiments.

Article Snippet: FITC-conjugated rat anti-mouse IgE (IgG1), FITC-rat IgG1 and FITC-conjugated rat anti-mouse CD117 (c-kit) were purchased from Cedarlane Laboratories (Burlington, ON, Canada).

Techniques: Mutagenesis, Derivative Assay, Staining, Flow Cytometry, Western Blot, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: The bidirectional effects of APPswe on the osteogenic differentiation of MSCs in bone homeostasis by regulating Notch signaling

doi: 10.1016/j.gendis.2024.101317

Figure Lengend Snippet: APPswe inhibits the self-renewal, proliferation, and migration of MSCs. (A, B) The effect of APPswe on the self-renewal ability of MSCs was detected by colony-forming unit-fibroblast assays. (C, D) Cell cycle changes in MSCs treated with APPswe were detected by flow cytometry. (E) The expression of the proliferation-related gene Ki67 in different groups was detected by cell immunofluorescence. (F) The expression levels of proliferation-related proteins in MSCs after treatment with APPswe were detected by western blotting. (G) Transwell assays were used to detect the effect of APPswe treatment on the cell migration ability of MSCs. (H) The effect of APPswe on MSC migration was detected by a wound healing test. (I) The expression levels of F-actin in the different treatment groups were detected by FITC-phalloidin staining. (J) Western blot analysis was performed to detect the expression levels of migration-related proteins in the different groups. n = 3; ∗ p < 0.05; ∗∗ p < 0.01, ∗∗∗ p < 0.001. APPswe, Swedish mutant amyloid precursor protein; MSC, mesenchymal stem cell.

Article Snippet: FITC anti-rat CD45, PE anti-rat CD90, and PE anti-rat CD29 antibodies were obtained from Elabscience Biotechnology (Wuhan, China).

Techniques: Migration, Flow Cytometry, Expressing, Immunofluorescence, Western Blot, Staining, Mutagenesis

Journal: Genes & Diseases

Article Title: The bidirectional effects of APPswe on the osteogenic differentiation of MSCs in bone homeostasis by regulating Notch signaling

doi: 10.1016/j.gendis.2024.101317

Figure Lengend Snippet: Effects of APPswe-C on the proliferation and migration of MSCs. (A, B) The effect of APPswe-C on the self-renewal of MSCs was detected by a colony-forming unit-fibroblast assay. (C, D) The effect of APPswe-C on the cell cycle was detected by flow cytometry. (E) The expression of the proliferation gene Ki67 was detected by cellular immunofluorescence. (F) The expression levels of proliferation-related proteins after APPswe-C treatment were detected by western blotting. (G, H) The cell migration of each group after APPswe-C treatment was detected by Transwell assay. (I, J) The effect of APPswe-C treatment on cell migration was detected by a wound healing test. (K) The expression of F-actin in the cells of each group after APPswe-C treatment was detected by FITC-phalloidin staining. (L) The expression levels of cell migration-related proteins were detected by western blot. MSCs were treated with APPswe-C for 24 h and then with BMP2 for 48 h to detect the expression levels of related proteins. n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. APPswe-C, Swedish mutant amyloid precursor protein without an intracellular segment; MSC, mesenchymal stem cell; BMP2, bone morphogenetic protein 2.

Article Snippet: FITC anti-rat CD45, PE anti-rat CD90, and PE anti-rat CD29 antibodies were obtained from Elabscience Biotechnology (Wuhan, China).

Techniques: Migration, Flow Cytometry, Expressing, Immunofluorescence, Western Blot, Transwell Assay, Staining, Mutagenesis

Journal: Arthritis Research & Therapy

Article Title: Human palatine tonsil: a new potential tissue source of multipotent mesenchymal progenitor cells

doi: 10.1186/ar2459

Figure Lengend Snippet: Surface epitope profile of tonsil-derived mesenchymal progenitor cells (T-MPCs). (a) Immunofluorescence analysis of cell surface epitope profiles of T-MPCs and bone marrow-derived mesenchymal progenitor cells (BM-MPCs). T-MPCs are shown in the top two rows of panels, and BM-MPCs are shown in the bottom two rows of panels. Epitopes were detected using fluorescently labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). Both cell populations were negative for CD14, CD34, and CD45 and positive for CD29, CD44, and CD105. Bars = 20 μm. (b) Flow cytometric analysis of T-MPCs and BM-MPCs. CD14, CD34, CD45, CD105, CD73, and CD90 were detected by fluorescently conjugated antibodies. The level of expression of each epitope is expressed as the mean fluorescence intensity ± standard deviation (n = 3). (c) Representative flow cytometry histogram. Control represents fluorescence due to the isotypic control. DAPI, 4',6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: For flow cytometry, T-MPCs (>1 × 10 5 cells) were washed and resuspended in PBS + 0.1% FBS (PF) containing saturating concentrations (1:100 dilution) of the following conjugated mouse IgG 1,κ anti-human monoclonal antibodies (BD Biosciences): HLA-A, B, C-phycoerythrin (PE) (MHC I), HLA-DR, DP, DQ-fluorescein isothiocyanate (FITC) (MHC II), CD45-FITC, CD14-PE, CD31-PE, CD34-PE, CD73-PE, CD90-FITC, CD105-PE as well as IFN-γR1-PE (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at 4°C.

Techniques: Derivative Assay, Immunofluorescence, Labeling, Staining, Expressing, Fluorescence, Standard Deviation, Flow Cytometry, Control

Journal: Nature immunology

Article Title: Mechanisms of CD40-dependent cDC1 licensing beyond costimulation

doi: 10.1038/s41590-022-01324-w

Figure Lengend Snippet: a, cDC1s were sorted from WT (Cd40+/+ CD45.2−, black) or Cd40−/− (CD45.2+, green) Flt3L-treated BM and cultured with CD8 T cells in the presence or absence of agonistic αCD40. Competitive survival was analyzed 48 h later. Numbers are percentages of cells in the indicated gates. b, Schematic diagram for experimental design used in panels c–f. Representative flow plot showing how cells can be distinguished by MHC-I and CD45.2 expression (cDC1s, MHC-I+ CD45.2+). Created with Biorender.com. c, Representative flow plots showing WT (top) and Cd40−/− (bottom) cDC1s. After 24 h culture as described in b, cells were incubated with FLICA and propidium iodide (PI), and cDC1 were analyzed by flow cytometry one hour later for PI and FLICA staining. Numbers are the percentages of cells in the indicated gates. Data represent two biologically independent experiments. d, Quantification of data shown in c at different cDC1:OT-I ratios (WT cDC1, black; Cd40−/− cDC1, green). Top, quantification of active caspase activity as a percent of MHC-I+ cDC1s. **P = 0.0016, ***P = 0.0001. Bottom, quantification of late apoptotic cells (FLICA+ Propidium iodide+) as a percent of MHC-I+ cDC1s. ****P < 0.0001. Data represent pooled independent samples from two biologically independent experiments (n = 5 for all groups). Data are represented as mean values ± s.d. e,f, Left graph shows representative two-color histograms of MitoTracker Deep Red FM (e) or MitoTracker Green FM (f) staining in WT (black) or Cd40−/− (green) cDC1s after 24 h culture described in panel b. Numbers are the geometric MFI of MitoTracker. Right graph shows data presented as individual samples. Data represent pooled independent samples from two biologically independent experiments (n = 9–10 for all groups). Data are represented as mean values ± s.d. ***P = 0.008. g,h, Extracellular flux analysis of cDC1s from WT (black), Cd40−/− (green) and Ptgs2cKO (red) Flt3L-treated BM. OCRs (g) or ECARs (h) before and after addition of pharmacological inhibitors are shown. Mean values of technical replicates ± s.e.m. of a representative experiment are shown. Plot representative of four biologically independent experiments. *P < 0.05; **P = 0.0020 and 0.0026; ***P = 0.0004. d–h, Two-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: The following antibody was used from Tonbo: CD45.1 (A20; 35-0453-U100).

Techniques: Cell Culture, Expressing, Incubation, Flow Cytometry, Staining, Activity Assay