Journal: Journal of leukocyte biology

Article Title: Pivotal advance: alpha-galactosylceramide induces protection against lipopolysaccharide-induced shock.

doi: 10.1189/jlb.0506298

Figure Lengend Snippet: Fig. 3. Cytokine production by spleen cells from LPS-KRN7000-treated mice before challenge and reexposed in vitro to -GalCer. C57BL/6 mice were injected with KRN7000 or with vehicle at indicated times before LPS challenge. 2 h after LPS challenge, groups of 10 mice were sacrificed. Pooled spleen cells from each group were cultured in complete medium with 10 ng/ml of KRN7000 (f) for 60 h at 37°C in 5% CO2 atmosphere. IFN (A), IL-4 (B), and IL-10 (C) were measured in culture supernatants by ELISA. Open triangles ( ) represent the levels of cytokines in mice treated with LPS-KRN7000 at the same times of previously showed experimental groups treated before challenge, and spleen cells were reexposed in vitro to vehicle. (A) Open circles () represent the levels of cytokines produced by splenocytes of mice treated with LPS and vehicle and reexposed in vitro to -GalCer. Results reported are the means of 3 different experiments. **, P 0.001.

Article Snippet: For measurement of cytokines levels, either in serum samples or in culture supernatants, we used IFN- , IL-4, IL-10, and TNF- Quantikine Mouse ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Produced

Journal: Journal of leukocyte biology

Article Title: Pivotal advance: alpha-galactosylceramide induces protection against lipopolysaccharide-induced shock.

doi: 10.1189/jlb.0506298

Figure Lengend Snippet: Fig. 2. Cytokines levels in sera of mice treated with KRN7000 before challenge. Groups of 10 mice were injected with KRN7000 or vehicle only from 1 to 18 h before LPS challenge. Sera were collected from 10 mice of each group 1 h after challenge for TNF- (A), 6 h after challenge for IFN- (B), and 2 h after challenge for IL-10 assay (C) (f). Data reported are the means of serum cytokines content tested for each animal of any group. Differences from cytokines levels detected in controls and experimental groups were reproduced without significant modifications when tested sera were collected at different time points following LPS challenge (data not shown). Serum cytokine levels were determined by ELISA. Data are the means of three different experiments. Before LPS challenge, the above cytokines were undetectable in the sera. Open triangles ( ) represent the values obtained from mice treated with LPS plus vehicle at the same times of experimental groups treated before challenge. In these control groups, sera were collected after challenge at the same times of experimental groups. **, P 0.001 *, P 0.05.

Article Snippet: For measurement of cytokines levels, either in serum samples or in culture supernatants, we used IFN- , IL-4, IL-10, and TNF- Quantikine Mouse ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of leukocyte biology

Article Title: Pivotal advance: alpha-galactosylceramide induces protection against lipopolysaccharide-induced shock.

doi: 10.1189/jlb.0506298

Figure Lengend Snippet: Fig. 4. KRN7000’s effects on systemic release of cytokines by mice challenged with LPS. Mice challenged with LPS were injected with KRN7000 or vehicle at indicated times after challenge. Sera of 10 mice for each experimental group were collected 4 h for TNF- (B), 6 h for IFN- (A), and 4 h for IL-10 (C) after challenge (f). Data reported are the means of serum cytokine content tested for each animal of any group. Open triangles ( ) represent the values obtained from sera of mice treated with LPS plus vehicle at the same times of experimental groups. Serum cytokine levels were determined by ELISA. Data represented are the means of 3 different experiments. Before LPS challenge, the above cytokines were undetectable in the sera. Differences from cytokine levels detected in controls and experimental groups were reproduced without significant modifications when sera tested were collected at different time points after LPS challenge (data not shown). **, P 0.001, *, P 0.05.

Article Snippet: For measurement of cytokines levels, either in serum samples or in culture supernatants, we used IFN- , IL-4, IL-10, and TNF- Quantikine Mouse ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Journal of leukocyte biology

Article Title: Pivotal advance: alpha-galactosylceramide induces protection against lipopolysaccharide-induced shock.

doi: 10.1189/jlb.0506298

Figure Lengend Snippet: Fig. 5. KRN7000 in vitro reexposure induces poor IFN but high IL-4 and IL-10 production by spleen cells from LPS challenged-GalCer-protected mice. C57BL/6 mice were injected at indicated times after LPS challenge with KRN7000 or with vehicle. One hour after -GalCer administration, groups of 10 mice were sacrificed. Pooled spleen cells from each group were cultured in complete medium with 10 ng/ml of KRN7000 (f) or vehicle ( ) for 60 h at 37°C in 5% CO2 atmosphere. IFN- (A), IL-4 (B) and IL-10 (C) were measured in cultured supernatants by ELISA assays. Open triangles represent cytokine levels in mice primed, challenged with LPS, treated with KRN7000 and reexposed in vitro to vehicle. (A) Open circles () represent the levels of cytokines produced by splenocytes of mice primed, challenged with LPS plus vehicle, and reexposed in vitro to -GalCer. Results reported are the means of 3 different experiments. **, P 0.001.

Article Snippet: For measurement of cytokines levels, either in serum samples or in culture supernatants, we used IFN- , IL-4, IL-10, and TNF- Quantikine Mouse ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Produced

Journal: Journal of leukocyte biology

Article Title: Pivotal advance: alpha-galactosylceramide induces protection against lipopolysaccharide-induced shock.

doi: 10.1189/jlb.0506298

Figure Lengend Snippet: Fig. 6. Cytokine production by NKT-negative and NK-negative fractions of protected and nonprotected mice. Spleen cells from KRN7000-protected (A) or nonprotected mice (B) were depleted of NKT and/or NK cells by immunomagnetic methods. NKT- and NK- fractions were obtained from protected or nonprotected mice. 4 105 of separated or unseparated cells were reexposed in vitro to -GalCer or vehicle dissolved in complete medium for 60 h at 37°C in 5% CO2 atmosphere. Cultured supernatants were tested for cytokines content by Quantikine ELISA kits. No significant difference in cytokines levels was detected testing both protected (treated with KRN7000 1.5 h before challenge or 1 h after challenge) or nonprotected groups (treated 18 h before challenge or 3 h after challenge). Data shown are the means of 3 different experiments.

Article Snippet: For measurement of cytokines levels, either in serum samples or in culture supernatants, we used IFN- , IL-4, IL-10, and TNF- Quantikine Mouse ELISA kits (R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay