Journal: STAR Protocols

Article Title: Assessing in vivo presentation of exogenous antigen in the tumor microenvironment in mice

doi: 10.1016/j.xpro.2023.102185

Figure Lengend Snippet:

Article Snippet: CD45.2 – vF450 (1:100) , Tonbo Biosciences , clone: 104; Cat# 65-0454-U100.

Techniques: Blocking Assay, Recombinant, Selection, Software, Cell Culture, Light Microscopy

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

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: IL-12 and IL-23 polarized CD4+ T cells are both capable of mediating ON. (A-C) C57BL/6 mice were immunized with MOG35-55 emulsified in CFA. Ten to fourteen days later, draining lymph node cells were harvested and cultured for 96 hours with MOG35-55 and either recombinant IL-12 or IL-23 to generate Th1 and Th17 cells, respectively. (A) Intracellular staining and flow cytometric analysis of cultured cells at 96 hours, gating on the CD3+CD4+ population. (B, C) Following culture, CD4+ T cells were transferred into naïve syngenic recipients. Mice in each group were euthanized at day 9 post-transfer, the day after clinical EAE onset. Mononuclear cells isolated from optic nerves (ON) and spinal cords (SC) were assessed for CD4+ T cell cytokine production (B) and the percent of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes (C) by flow cytometry. Data are representative of three independent experiments with at least 3 mice per group.

Article Snippet: Antibodies and reagents The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Cell Culture, Recombinant, Staining, Isolation, Flow Cytometry

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

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Axonopathy is an early feature of both Th1- and Th17-mediated ON. (A–D) Th1 and Th17 adoptive transfer recipients were injected i.o. with fluorochrome-conjugated cholera toxin-B (red) at onset of clinical EAE. Optic nerves were harvested 24 hours later. Longitudinal sections were stained for the pan-leukocyte marker, CD45 (green) to demarcate areas of inflammation. (C) Confocal microscopy revealed a swelling at the tip of a transected axon (asterisk). (D) The frequency of axonal swellings was compared with the intensity of CD45 staining and there was no significant difference. (E–G) Cross- sections of optic nerves harvested from mice with Th17- (E) and Th1- (F) mediated ON were stained for unphosphorylated neurofilament-H (SMI-32, green; DAPI, blue; bars=50 μm). Magnified views of the insets are shown in adjacent panels (bars=25 μm). (G) SMI-32 staining of a healthy nerve (bar= 50 μm). (H–J) Representative cross-sections of optic nerves obtained from mice with Th17- (H) or Th1-(I) mediated ON, or from naïve mice (J), were stained with toludine blue. Broken lines delineate representative areas with normal-appearing axons, asteriscs indicate swollen axons and arrowheads point to empty myelin sheaths (bars= 25 μm).

Article Snippet: Antibodies and reagents The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Adoptive Transfer Assay, Injection, Staining, Marker, Confocal Microscopy

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

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Axon loss and faulty nerve conduction occur in both Th1- and Th17-mediated ON. (A–D) Optic nerves harvested immediately before the peak of EAE were stained with antibodies against SMI32 (green) and CD45 (red). Representative cross-sections of nerves from mice with Th17- (A) and Th1- (B) mediated ON, as well as from a naïve mouse (B), are shown (bars = 100 μm and 25 μm, inset) and quantified (D). (E) Brn3a+ RGCs were counted in retinas harvested from naïve mice, and from ON mice at serial time points post-transfer (t-test). (F) Representative traces of CAPs of optic nerves acutely isolated from naïve and ON mice. CAP velocities (G, I) and amplitudes (H, J) were averaged over seven to twelve nerves per group. Data presented as mean ±S.E.M. (**p<0.01, ***p<0.001, ****p<0.0001; Mann-Whitney) Scale bars: A, B 50μm, insets 25μm; C 25μm.

Article Snippet: Antibodies and reagents The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Staining, Isolation, MANN-WHITNEY

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

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Stable Th17 cells induce ON. (A) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-12KO mice, after 96 hours of culture with MOG35-55 and recombinant IL-23. (B) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-12KO donors after infiltrating the spinal cords and optic nerves of IL-12KO hosts. (C) Clinical courses of IL-12KO mice injected with myelin-specific CD4+ Th17 cells derived from IL-12KO donors versus WT mice injected with WT effector Th17 cells. The data shown is pooled from 3 experiments with n=10 WT and n=17 IL12KO host mice per group. (D) The percentage of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes infiltrating the optic nerve and spinal cord of IL-12KO hosts shortly after the onset of clinical EAE. (E) Contiguous sections of an optic nerve obtained from an IL-12KO host at clinical EAE onset and stained with CD45 (red, top left panel) or SMI-32 (green). (F) A representative section stained with toludine blue (bars= 50μm in D, 25μm in E). Areas with normal appearing axons are outlined, asteriscs indicate swollen axons and arrowheads point to empty myelin sheaths. (G–I) CAPs were measured in acutely isolated optic nerves from the IL-12KO recipients of stable Th17 cells at clinical EAE onset. (G) Representative wave-forms of optic nerve CAPs from a naïve IL-12KO mouse and an IL-12KO adoptive transfer recipient with acute ON. The data were averaged over seven to nine nerves per group. Data are presented as mean ±S.E.M. (*p<0.05, **p<0.01; Mann-Whitney)

Article Snippet: Antibodies and reagents The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Derivative Assay, Recombinant, Injection, Staining, Isolation, Adoptive Transfer Assay, MANN-WHITNEY

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

Article Title: T-helper cell diversity in experimental autoimmune encephalomyelitis and Multiple Sclerosis

doi: 10.4049/jimmunol.1501097

Figure Lengend Snippet: Bona Fide Th1 cells are capable of inducing ON. (A) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-23KO mice, after 96 hours of culture with MOG35-55 and recombinant IL-12. (B) Intracellular cytokine production by CD4+ T cells derived from MOG-immunized IL-23KO donors after infiltrating the spinal cords and optic nerves of IL-23KO hosts. (C) Clinical courses of IL-23KO mice injected with myelin-specific CD4+ Th1 cells derived from IL-23KO donors versus WT mice injected with WT effector Th17 cells. The data was pooled from 3 experiments with n=9 WT and n=22 IL-23KO hosts (*p<0.05, **p<0.01; Holm-Sidak multiple t-tests). (D) The percentage of CD4+ T cells and CD11b+ myeloid cells among CD45+ leukocytes infiltrating the optic nerve and spinal cord. (E) Contiguous sections of a representative optic nerve obtained from an IL-23KO host at clinical EAE onset and stained with CD45 (red, left panel) or SMI-32 (green, right panels). (F) A representative section stained with toluidine blue. (bars= 50μm in E, 25μm in insets and in F). Examples of areas with normal appearing axons are outlined, asterisks mark examples of swollen axons and arrowheads point to myelin sheaths left behind by degenerated axons. (G–I) CAPs were measured in optic nerves from the IL-23KO recipients of Th1 cells at clinical EAE onset. The data were averaged over seven to nine nerves per group. (G) Representative wave-forms. CAP velocities (H) and amplitudes (I) were measured in eight to twelve nerves per group. Data are presented as mean ± S.E.M. (*p<0.05, **p<0.01; Mann-Whitney)

Article Snippet: Antibodies and reagents The following antibodies were used for flow cytometry or immunohistochemistry: rat anti-MBP (Millipore); mouse anti-unphosphorylated neurofilament-H (SMI-32, Covance); CD45 (Serotec), Brn3a (Santa Cruz); Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rat IgG (Life Technologies); FITC–anti-MHCII, FITC–anti-B220, PE–anti-CD45 (Ly5), PE–anti-CD8α, PE–anti-CD4, PE–anti–GM-CSF, PECy7–anti-CD11b, and PECy7–anti-CD4; PerCpCy5.5–anti-Ly6C, PerCPCy5.5–anti-CD3ε, and PerCPCy5.5–anti–IL-17A (e-Bioscience); BD Biosciences: allophycocyanin–anti-CD45.2, FITC–anti-CD44, allophycocyanin cy7–anti-Ly6G, allophycocyanin cy7–anti-CD45.1, and allophycocyanin cy7–anti–IFN-γ (BD Biosciences).

Techniques: Derivative Assay, Recombinant, Injection, Staining, MANN-WHITNEY

Journal: iScience

Article Title: The ATP-exporting channel Pannexin 1 promotes CD8 + T cell effector and memory responses

doi: 10.1016/j.isci.2024.110290

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Article Snippet: vf450 anti-mouse CD45.1 , Cytek/Tonbo Biosciences , Cat# SKU 75-0451-U025; RRID:AB_2621947.

Techniques: Purification, Virus, Recombinant, Blocking Assay, Saline, Cell Stimulation, Cell Isolation, Isolation, Infection, In Vitro, Software

Journal: Frontiers in Pharmacology

Article Title: Jianpi Yangzheng Xiaozheng decoction alleviates gastric cancer progression via suppressing exosomal PD-L1

doi: 10.3389/fphar.2023.1159829

Figure Lengend Snippet: JPYZXZ reduced the number of MDSCs in tumor bearing mice. (A,B) Percentage of MDSCs (CD45 + CD11b+Gr1+) in blood determined using flow cytometry. (C,D) Percentage of MDSCs (CD45 + CD11b+Gr1+) in tumors determined using flow cytometry. (E) Immunofluorescence staining for DAPI (nucleus), CD11b, Gr-1, and PD-L1 in tumor tissue sections from the xenograft tumor model. Data are represented as the mean ± SEM, n = 5 mice or n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The single-cell suspensions (1 × 10 7 cells/mL) were stained with fluorochrome-coupled antibodies AF488-CD45 (Elabscience, E-AB-F1136L), PE/Dazzle™594-CD11b (BioLegend, 101256), and APC-Gr-1 (BioLegend, 108412) for 30 min at 4°C.

Techniques: Flow Cytometry, Immunofluorescence, Staining

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