goat polyclonal anti-gst3 / gst pi antibody  (Advisains)

Journal: The Journal of Experimental Medicine

Article Title: Type I interferons drive MAIT cell functions against bacterial pneumonia

doi: 10.1084/jem.20230037

Figure Lengend Snippet: Type I IFN–dependent MAIT cell activation. (A) Murine MAIT cells were sorted from the lungs of WT mice (previously injected with 5-OP-RU+LPS to expand the MAIT cell population) and cultured with IFNα or the supernatant of Klebsiella -treated RAW264.7 cells. Representative flow cytometry plots (from three independent experiments) show phosphorylation of STAT1 in MAIT cells at the indicated time points after stimulation with IFNα (top) or the supernatant of Klebsiella -treated RAW264.7 cells (bottom). Red profile = stimulated cells; blue = unstimulated controls. (B) MAIT cells were sorted from the lungs of WT mice (previously injected with 5-OP-RU+LPS to expand the MAIT cell population) and cultured (for 18 h) with RAW264.7 macrophages in the presence/absence of WT (KP, Kp43816) or mutant KPΔ ribD as indicated. Frequencies of Granzyme B (GrzB + ), IFN-γ + , IL-17 + , or CD69 + MAIT cells is shown; data are pooled from two independent experiments with MAIT cells obtained from three to five mice. Bars represent mean ± SEM, *P < 0.5, ***P < 0.001, ****P < 0.0001; ns, not significant, one-way ANOVA with Tukey’s multiple comparisons test. (C) Flow cytometry plots and quantification of MAIT cell frequency in WT mice after i.n. challenges with PBS (blue), LPS (black), and 5-OP-RU+LPS (red). Lines represent mean ± SEM, each dot represents a mouse ( n = 5–20). **P < 0.01, one-way ANOVA with Tukey’s multiple comparisons test. (D) Flow cytometry plots showing T-bet/RORγt, CD4/CD8, CD69/CD25/CD137, IFNγ/IL-17A/GramB in pulmonary MAIT cells from PBS-treated (control) vs. 5-OP-RU/LPS-treated mice. (E) Flow cytometry plots for CXCR6 expression in MAIT cells (CD3ε + MR1(5-OP-RU)Tetramer + ), invariant natural killer T (iNKT) cells (CD3ε + CD1d(PBS-57)Tetramer + ), and γδT cells (CD3ε + TCRγδ + ) in the lungs of WT mice. (F) Flow cytometry plots showing CD3 + GFP + cells and frequency of MAIT cells, iNKT cells, and γδ T cells within the CD3 + GFP + population in the lung of CXCR6-eGFP mice without (top) or with (bottom) 5-OP-RU+LPS administration.

Article Snippet: Samples were stained with rabbit polyclonal αCD3ε antibody (Abcam), αPodoplanin/gp36 (PMab-1; Abcam), and goat polyclonal αGFP (Abcam) antibodies followed by donkey anti-rabbit IgG Alexa Fluor Plus 594 (Invitrogen), donkey anti-rat IgG DyLight 680 (Invitrogen), and donkey anti-goat IgG Alexa Fluor 488 (Invitrogen).

Techniques: Activation Assay, Injection, Cell Culture, Flow Cytometry, Phospho-proteomics, Mutagenesis, Control, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Type I interferons drive MAIT cell functions against bacterial pneumonia

doi: 10.1084/jem.20230037

Figure Lengend Snippet: K. pneumoniae infection induces MAIT cell relocation in the lung tissue. (A and B) WT mice were infected with K. pneumoniae or received PBS (−KP, control) and were i.v. injected with FITC-labeled αCD45 antibody 3 min prior to tissue collection. (A) Representative flow cytometry plots (middle) and quantification (right) of the frequency of pulmonary MAIT cells stained (CD45 + ) or not (CD45 − ) with FITC-labeled αCD45. (B) Representative flow cytometry plots (left) and quantification (right) of the frequency of CD69 + pulmonary MAIT cells stained (CD45 + ) or not (CD45 − ) with FITC-labeled αCD45. Lines represent mean ± SEM, each dot is a mouse ( n = 5), and data are pooled from two to three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. (C) Left: Staining of lung tissue (FLASH) for CXCR6-GFP mice (previously injected with 5-OP-RU+LPS to expand the MAIT cell population) showing GFP (pink), CD3 (yellow), and podoplanin (blue). Arrowheads indicate MAIT cells identified as CD3 + GFP + cells. Mice were infected with K. pneumoniae (+KP) or received PBS (−KP) and αIFNAR blocking antibody (bottom) or isotype control (top) prior to infection. Scale bar = 50 μm. Right: Quantification of cell distance evaluated by measuring the spatial distance (μm) between CD3 + GFP + cells. Each dot represents the average distance (μm) between all CD3 + GFP + cells per frame ( n = 9–18 frames per condition) from two independent experiments. *P < 0.05, **P < 0.01, two-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Samples were stained with rabbit polyclonal αCD3ε antibody (Abcam), αPodoplanin/gp36 (PMab-1; Abcam), and goat polyclonal αGFP (Abcam) antibodies followed by donkey anti-rabbit IgG Alexa Fluor Plus 594 (Invitrogen), donkey anti-rat IgG DyLight 680 (Invitrogen), and donkey anti-goat IgG Alexa Fluor 488 (Invitrogen).

Techniques: Infection, Control, Injection, Labeling, Flow Cytometry, Staining, Blocking Assay