Journal: Science Advances

Article Title: Altered X-chromosome inactivation predisposes to autoimmunity

doi: 10.1126/sciadv.adn6537

Figure Lengend Snippet: ( A ) Representative flow cytometry analysis of spontaneously activated B220 + CD69 + B cells in spleen from 1-year-old WT and Ftx −/− females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown (Mann-Whitney test, * P < 0.05 and ** P < 0.01). ( B ) Same as (A) for spontaneously activated CD4 + CD69 + T cells (Mann-Whitney test, * P < 0.05 and ** P < 0.01). ( C ) Total IgG, IgM, IgG2b, and IgG2c natural antibody levels in sera of 3-month-, 1-year, and >1.5-year-old WT or Ftx −/− females measured by ELISA. Each circle represents a mouse. Mean values are shown (Mann-Whitney test, * P < 0.05 and ** P < 0.01). ( D ) Cytokines levels in the blood analyzed with cytometric bead array assays on sera from 3-month-, 1-year-, or 2-year-old WT and Ftx −/− females. Each triangle represents a mouse. Median values are shown ( t test, * P < 0.05).

Article Snippet: BM, spleen, blood, and peritoneal cavity cells were stained using the following antibodies: CD3 PerCP-Vio770 (130-119-656, Miltenyi Biotec), CD4-allophycocyanin (APC) (130-123-207, Miltenyi Biotec), CD5-APC-Vio770 (130-120-165, Miltenyi Biotec), CD8-fluorescein isothiocyanate (FITC) (130-118-468, Miltenyi Biotec), CD11b APC (553312, BD Pharmingen), CD11c phycoerythrin (PE)–Vio770 (130-110-840, Miltenyi Biotec), CD19-FITC (557398, BD Pharmingen), CD21-APC-Vio770 (130-111-733, Miltenyi Biotec), CD23-PE-Vio770 (130-118-764, Miltenyi Biotec), CD38-PE (130-123-571, Miltenyi Biotec), CD43-PE (130-112-887, Miltenyi Biotec), CD69-PE (130-115-575, Miltenyi Biotec), CD138 PE-Vio615 (130-108-989, Miltenyi Biotec), F4/80 FITC (130-117-509, Miltenyi Biotec), Ter119 PE (130-112-909, Miltenyi Biotec), SiglecH APC-Vio770 (130-112-299, Miltenyi Biotec), B220-APC (130-110-847, Miltenyi Biotec), B220 VioBlue (130-110-851, Miltenyi Biotec), IgM-VioBlue (130-116-318, Miltenyi Biotec), IgD-PE (130-111-496, Miltenyi Biotec), GL7-PE-Cy7 (144619, BioLegend), Ly6C-FITC (130111-915, Miltenyi Biotec), streptavidin FITC (554060, BD Biosciences), CD138 BV605 (563147, BD-Horizon), CD23 BV605 (101637, BioLegend), I-A/I-E BV711 (107643, BioLegend), CD19 BV786 (563333, BD Horizon), T and B cell activation antigen (GL7) PE (561530, BD Pharmingen), CD95 PE-Cy7 (557653, BD Pharmingen), IgM APC-eFluor 780 (47-5790-82, Invitrogen), CD45R/B220 APC/cyanine7 (103224, BioLegend), CD11b eFluor 450 (48-0112-82, eBioscience), CD267 (TACI) BV421 (742840, BD Biosciences), IgD BUV395 (564274, BD Horizon), streptavidin APC (4317-82, eBioscience), CD3 Biotin (100304, BioLegend), Biotin CD11c (568970, BD Biosciences), CD21 PercP Cy5.5 (562797, BD Biosciences), and Fixable Viability Dye eFluor 506 (65-0866-18, Invitrogen) following the recommendations of the manufacturers.

Techniques: Flow Cytometry, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Killing of Latently HIV-Infected CD4 T Cells by Autologous CD8 T Cells Is Modulated by Nef

doi: 10.3389/fimmu.2018.02068

Figure Lengend Snippet: Resting CD4 T cells procured from HIV infected patients on ART. (A) Representative analysis indicating the purity of resting CD4 T cells. Resting CD4 T cells procured from the PBMC of HIV infected patients on ART were purified using a two-step negative selection, stained with anti-human CD25, anti-human CD69 and anti-human HLA-DR antibodies and analyzed by the ImageStream. (B) The fraction of resting and activated CD4 T cells. CD4 T cells procured from the PBMC of HIV infected patients on ART were labeled with antibodies against CD25, CD69 and HLA-DR followed by ImageStream analysis. 51% of the CD4 T cells were CD25, CD69, and HLA-DR positive (activated cells) and 49% were CD25, CD69, and HLA-DR negative (resting cells).

Article Snippet: Resting CD4 T cells were separated from activated CD4 T cells using a two-step negative selection method based on the activated cells surface markers CD25, CD69, and HLA-DR, ( – ) using CD69 MicroBead kit II, Anti-HLA-DR MicroBeads, and anti-human CD25 conjugated to microbeads (MicroBeads II, Miltenyi Biotech) according to the manufacturer's protocol (Miltenyi Biotech).

Techniques: Infection, Purification, Selection, Staining, Labeling

Journal: Frontiers in Immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: JHU083 inhibited T cells activation in vivo . Mice of Vehicle-AIH and JHU083-AIH group were intravenously injected with 8ml/kg ConA, and Vehicle-WT group was injected with normal saline of equal volume. JHU083-AIH group was gavaged with 0.3mg/kg JHU083 24h and 1h before ConA injection, and mice of Vehicle-WT and Vehicle-AIH group were treated with equal vehicle at the same time points. (A, B) Flow cytometry was applied to analyze the expression of CD25 and CD69 of CD4(+) (A) and CD8(+) (B) T cells. Data were expressed as means ± SEM. * P < 0.05 and ** P <0.01.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PE-Cy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNγ (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: Activation Assay, In Vivo, Injection, Saline, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: DON treatment suppressed T cells activation in vitro . Freshly separated spleen cells were stimulated with 1.5mg/ml ConA with or without treating 2.5μM DON, and cells were analyzed 24h after ConA stimulation. (A, B) The expressions of activation markers CD25 and CD69 in CD4(+) (A) and CD8(+) (B) T cells were examined by flow cytometry. Data were expressed as means ± SEM. ** P <0.01, and *** P <0.001.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PE-Cy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNγ (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: Activation Assay, In Vitro, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation

doi: 10.3389/fimmu.2022.880262

Figure Lengend Snippet: The function of DON to mTOR signaling may be mediated by amino transporter amino transporter SLC7A5. (A) qRT-PCR was applied to analyze amino acid transporters SLC7A5 and SLC1A5 mRNA levels. (B, C) Flow cytometry was applied to analyze the degree of activation (expression of CD25 and CD69) of CD4(+) (B) and CD8(+) (C) T cells with or without treating LAT1-IN1. The levels of phosphorylated mTOR (D) as well as phosphorylated P70S6K (E) were examined by western blotting. Data were expressed as means ± SEM. *P < 0.05, **P <0.01, and ***P <0.001. ns, not significant.

Article Snippet: The antibodies used in the flow cytometry were as follows: CD4 (FITC, E-AB-F1097C), CD8a (PE, E-AB-F1104D), CD25 (PE-Cy5, E-AB-F1102G), CD69 (PE-Cy7, E-AB-F1187H), IL4 (PE, E-AB-F1204UD), IL17A (APC, E-AB-F1272E) were purchased from Elabscience Biotechnology (Wuhan, China), antibodies IFNγ (APC-Cy7, cat# 561479) was purchased from BD Biosciences (UK), and Granzyme B (PE-Cy7, cat# 372213) was purchased from Biolegend (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Flow Cytometry, Activation Assay, Expressing, Western Blot