il 17a mouse duo set elisa  (R&D Systems)

Journal: Mucosal Immunology

Article Title: Generation of protective pneumococcal-specific nasal resident memory CD4 + T cells via parenteral immunization

doi: 10.1038/s41385-019-0218-5

Figure Lengend Snippet: Pneumococcal-responsive IL-17A + CD4 + nasal T RM are detected after I.N. exposure. The nasal mucosa (NM) and spleen were harvested from mice 1–2 months after I.N. inoculation with serotype 6B pneumococcal strain 0603 or sham infection with PBS. a Representative dot plot showing intracellular staining for IL-17A in CD4 + T cells following ex vivo stimulation of NM and splenic cells with PMA/ionomycin (P/I) or pneumococal whole-cell antigen (WCA). Quantitation of frequency of IL-17A + CD4 + T cells after ( b ) P/I or ( c ) WCA stimulation. d Absolute number of IL-17A + CD4 + T cells in the nose and spleen after stimulation with WCA. The results represent a pool of three experiments, n = 2–5 mice/group/experiment. Horizontal lines indicate the median value. * P < 0.05, ** P < 0.01. *** P < 0.001, **** P < 0.0001

Article Snippet: Supernatants were harvested and IL-17A was detected in the supernatants by ELISA as per the mouse IL-17A ELISA DUO set kit instructions (reference DY421; R&D Systems).

Techniques: Infection, Staining, Ex Vivo, Quantitation Assay

Journal: Mucosal Immunology

Article Title: Generation of protective pneumococcal-specific nasal resident memory CD4 + T cells via parenteral immunization

doi: 10.1038/s41385-019-0218-5

Figure Lengend Snippet: Nasal T RM generated after exposure to live pneumococcus are sufficient to mediate protection from secondary challenge. Mice were exposed to live pneumococcus (0603) or not (PBS), 1 month later treated with FTY720 (+FTY) or left untreated, then challenged I.N. with 0603. Nasal washes and tissue harvest were performed 7 days post challenge. a CFU in nasal washes. b Frequency and (c) absolute number of IL-17A + CD4 + T cells in the nose after ex vivo stimulation with WCA for 24 h. d Concentration of IL-17A in supernatants from nasal cells after ex vivo stimulation with WCA for 7 days. a represents a pool of four experiments, n = 5–10 mice/group/experiment. b – d represent a pool of two experiments, n = 5 mice/group/experiment. Horizontal lines indicate the median value. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Supernatants were harvested and IL-17A was detected in the supernatants by ELISA as per the mouse IL-17A ELISA DUO set kit instructions (reference DY421; R&D Systems).

Techniques: Generated, Ex Vivo, Concentration Assay

Journal: Mucosal Immunology

Article Title: Generation of protective pneumococcal-specific nasal resident memory CD4 + T cells via parenteral immunization

doi: 10.1038/s41385-019-0218-5

Figure Lengend Snippet: CD4 + T RM generated after mucosal immunization with WCV are sufficient to provide protection following nasal challenge with pneumococcus. Nasal washes and nasal tissue harvest were performed 4–7 days after challenge of mice immunized with CT or WCV and then left untreated, treated with FTY720, or anti-CD4 antibody (as indicated). a Levels of nasal colonization with pneumococcus. The results are representative of two experiments, n = 10 mice/group. b Absolute number of CD69 + CD11a + CD4 + T m cells in the nose. Frequency ( c ) and absolute number ( d ) of IL-17A + CD4 + T cells in the nose after ex vivo stimulation with WCA for 24 h. e Concentration of IL-17A in supernatants from nasal cells after ex vivo stimulation with WCA for 7 days. The results represent a pool of two experiments, n = 4–5 mice/group/experiment. Horizontal bars indicate the median value. * P ≤ 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Supernatants were harvested and IL-17A was detected in the supernatants by ELISA as per the mouse IL-17A ELISA DUO set kit instructions (reference DY421; R&D Systems).

Techniques: Generated, Ex Vivo, Concentration Assay

Journal: Mucosal Immunology

Article Title: Generation of protective pneumococcal-specific nasal resident memory CD4 + T cells via parenteral immunization

doi: 10.1038/s41385-019-0218-5

Figure Lengend Snippet: Pneumococcus-responsive CD4 + T RM are detected in the nose and colon after parenteral immunization. Cells isolated from the NM, spleen, or colon from groups of mice immunized with alum alone or WCV were analyzed by flow cytometry, either after staining with antibodies to identify CD4 + T RM , or 5 h after stimulation with PMA/Ionomycin (P/I) or 24 h after stimulation with WCA. a Absolute number of CD69 + CD11a + CD4 + T m cells in each tissue. b Frequency of CD4 + T cells staining with IL-17A following stimulation with PMA/ionomycin or WCA. c Absolute numbers of IL-17A + CD4 + T cells after stimulation with WCA. d Frequency of CD4 + T cells staining with IFNγ following stimulation with PMA/ionomycin or WCA. The results represent a pool of two experiments, n = 3–5 mice/group/experiment. Horizontal bars indicate the median value. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Supernatants were harvested and IL-17A was detected in the supernatants by ELISA as per the mouse IL-17A ELISA DUO set kit instructions (reference DY421; R&D Systems).

Techniques: Isolation, Flow Cytometry, Staining

Journal: Mucosal Immunology

Article Title: Generation of protective pneumococcal-specific nasal resident memory CD4 + T cells via parenteral immunization

doi: 10.1038/s41385-019-0218-5

Figure Lengend Snippet: WCV-induced CD4 + T RM generated after parenteral immunization provide long-lived protection and are generated early in life after a single dose. Mice immunized with alum alone or WCV were left untreated, treated with FTY720, or treated with anti-CD4 antibody as indicated, challenged I.N. with pneumococcus, and analyzed 10 days later. a Levels of nasal colonization with pneumococcus. b Concentration of IL-17A in supernatants from whole-blood samples 6 days post ex vivo stimulation with WCA. c Five-week-old female mice were immunized three times S.C. with alum or WCV, 9–22 months later mice were left untreated, or treated with FTY720, challenged I.N. with 0603 and levels of colonization were measured 10 days later. d Mice were immunized once S.C. at 6 days of life (DOL 6) with Alum or WCV. Eight weeks later, mice were left untreated, or treated with FTY720 as indicated, challenged I.N. with 0603 and levels of colonization were measured 10 days later. The results in ( a and b ) are a representative experiment from two experiments, n = 8–10 mice/group. The results in ( c and d ) represent a pool of two experiments, n = 7–10 mice/group/experiment. Horizontal bars indicate the median value. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Supernatants were harvested and IL-17A was detected in the supernatants by ELISA as per the mouse IL-17A ELISA DUO set kit instructions (reference DY421; R&D Systems).

Techniques: Generated, Concentration Assay, Ex Vivo