extraction solvent contained c17 ceramide  (Croda International Plc)

Journal: eLife

Article Title: Cell-intrinsic ceramides determine T cell function during melanoma progression

doi: 10.7554/eLife.83073

Figure Lengend Snippet: Isolated CD8 + T cells from Smpd1 fl/fl /Cd4 cre/+ mice (Asm/CD4cre) and Smpd1 fl/fl /Cd4 +/+ littermates (wildtype [WT]) where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. ( A ) mRNA expression of Asm ( Smpd1 ) following activation was analyzed by RT-qPCR (n=4–8). ( B ) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n=4). ( C ) Expression of CD25, CD69, and CD44 was analyzed by flow cytometry (n=6–7). Representative histograms are shown in the upper panel. ( D ) OVA-specific cytotoxic lymphocytes were generated and incubated with OVA-peptide 257–264 loaded CFSE high -labeled target and unloaded CFSE low -labeled control cells. Specific killing was evaluated by frequencies of target and control populations determined by flow cytometry (n=6–7). Representative histograms are shown in the left panel. ( E ) Frequencies of granzyme B-expressing CD8 + T cells from Asm/CD4cre mice and WT littermates without and ( F ) in the presence of C16 ceramide were analyzed by flow cytometry (n=4–8). Representative contour plots are shown in the left panel. Results from two to four independent experiments are depicted as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test, or Student’s t-test. (*p<0.05, **p<0.01, ****p<0.0001). Figure 3—source data 1. T cell-specific Asm deficiency leads to reduced CD8 + T cell activation in vitro.

Article Snippet: The extraction solvent contained C17 ceramide (C17 Cer) and d 7 -S-1-P (both Avanti Polar Lipids, Alabaster, USA) as internal standards.

Techniques: Isolation, Expressing, Activation Assay, Quantitative RT-PCR, Mass Spectrometry, Flow Cytometry, Generated, Incubation, Labeling, MANN-WHITNEY, In Vitro

Journal: eLife

Article Title: Cell-intrinsic ceramides determine T cell function during melanoma progression

doi: 10.7554/eLife.83073

Figure Lengend Snippet: ( A ) Isolated CD4 + T cells from Asm/CD4cre mice and wildtype (WT) littermates were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for indicated time points. mRNA expression of Asm ( Smpd1 ) was analyzed by RT-qPCR (n=6–8). ( B ) Ceramide levels of unstimulated or anti-CD3/anti-CD28 stimulated (24 hr) CD4 + T cells from Asm/CD4cre mice and WT littermates were determined by mass spectrometry (n=4). ( C ) Sorted CD4 + CD25 − T cells from Asm/CD4cre and WT mice were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and TGF-β1 (iTreg). Respective controls (Th0) were only stimulated with anti-CD3 and anti-CD28 antibodies. After 3 days, Treg differentiation was analyzed by Foxp3 expression (n=6–7). Representative dot plots are depicted in the left panel. ( D ) In order to differentiate naïve CD4 + CD25 − T cells into Th1 cells, isolated cells were cultured in the presence of anti-CD3, anti-CD28, anti-IL-4, and IL-12 (Th1), or only stimulated with anti-CD3 and anti-CD28 (Th0). After 6 days, Th1 differentiation was assessed by IFN-γ expression (n=5–6). Representative dot plots are shown in the left panel. Results from two to three independent experiments are depicted as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p<0.05, **p<0.01, ****p<0.0001).

Article Snippet: The extraction solvent contained C17 ceramide (C17 Cer) and d 7 -S-1-P (both Avanti Polar Lipids, Alabaster, USA) as internal standards.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Mass Spectrometry, Cell Culture

Journal: eLife

Article Title: Cell-intrinsic ceramides determine T cell function during melanoma progression

doi: 10.7554/eLife.83073

Figure Lengend Snippet: CD8 + T cells were isolated and stimulated with CD3/CD28 MACSiBead particles for 2 hr and stained for ceramide (red) and ( A ) CD3 or ( B ) TCR beta (green). Cells were visualized using a Biorevo BZ-9000 fluorescence microscope.

Article Snippet: The extraction solvent contained C17 ceramide (C17 Cer) and d 7 -S-1-P (both Avanti Polar Lipids, Alabaster, USA) as internal standards.

Techniques: Isolation, Staining, Fluorescence, Microscopy

Journal: eLife

Article Title: Cell-intrinsic ceramides determine T cell function during melanoma progression

doi: 10.7554/eLife.83073

Figure Lengend Snippet: ( A ) Isolated CD8 + T cells from Asah1 fl/fl /Cd4 cre/+ mice (Ac/CD4cre) and Asah1 fl/fl /Cd4 +/+ littermates (wildtype [WT]) where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. mRNA expression of Ac ( Asah1 ) following activation was analyzed by RT-qPCR (n=3–4). ( B ) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n=4). ( C ) For T cell receptor signaling analysis, splenocytes from Ac/CD4cre and WT mice were left unstimulated (0’) or stimulated with anti-CD3 and anti-CD28 for 5 (5’) min. Afterward, samples were analyzed for phospho-ZAP70 of gated ZAP70 + CD8 + T cells and phospho-PLCγ of gated CD8 + T cells by flow cytometry (n=4). Representative dot plots and fluorescence minus one (FMOs) for phospho-ZAP70 are shown in the left panel. ( D ) Western blot analysis of phospho-ZAP70 expression of CD8 + T cells from Ac/CD4cre and WT mice after 5 min of stimulation with anti-CD3 and anti-CD28 (n=3). ( E ) CD8 + T cells were left untreated as control or stimulated for 24 or 48 hr and analyzed for granzyme B expression by flow cytometry (n=5–8). Representative contour plots are shown in the left panel. ( F ) Specific killing of antigen-specific cytotoxic lymphocytes from Ac/CD4cre/OTI mice and WT controls was assessed (n=8–9). Representative histograms are shown in the left panel. Data are depicted as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test, or Student’s t-test. (*p<0.05, **p<0.01, ****p<0.0001). Figure 6—source data 1. Ac-deficient CD8 + T cells have elevated ceramide levels and show increased activation in vitro.

Article Snippet: The extraction solvent contained C17 ceramide (C17 Cer) and d 7 -S-1-P (both Avanti Polar Lipids, Alabaster, USA) as internal standards.

Techniques: Isolation, Expressing, Activation Assay, Quantitative RT-PCR, Mass Spectrometry, Flow Cytometry, Fluorescence, Western Blot, MANN-WHITNEY, In Vitro

Journal: eLife

Article Title: Cell-intrinsic ceramides determine T cell function during melanoma progression

doi: 10.7554/eLife.83073

Figure Lengend Snippet: ( A ) Isolated CD4 + T cells from Ac/CD4cre and wildtype (WT) mice were either left unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for indicated time points. mRNA expression of Ac ( Asah1 ) was analyzed by RT-qPCR (n=3–4). ( B ) Ceramide levels of unstimulated and anti-CD3/anti-CD28 stimulated (24 hr) CD4 + T cells from Ac/CD4cre and WT mice were determined by mass spectrometry (n=4). ( C ) For T cell receptor signaling analysis, splenocytes from Ac/CD4cre and WT mice were left unstimulated (0‘) or stimulated with anti-CD3 and anti-CD28 antibodies for 5 (5’) min. Afterward, samples were analyzed for phospho-ZAP70 of gated ZAP70 + CD4 + T cells and phospho-PLCγ of gated CD4 + T cells by flow cytometry (n=4–7). Representative dot plots and FMOs are shown in the left panel. ( D ) In order to differentiate naïve CD4 + CD25 − T cells from Ac flox/flox /CD4cre and WT mice into Th1 cells, isolated cells were cultured in the presence of anti-CD3, anti-CD28, anti-IL-4, and IL-12 (Th1), or only stimulated with anti-CD3 and anti-CD28 antibodies (Th0). After 6 days, cells were analyzed for IFN-γ and granzyme B expression by flow cytometry (n=9). Results from two to three independent experiments are depicted as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p<0.05, **p<0.01, ****p<0.0001).

Article Snippet: The extraction solvent contained C17 ceramide (C17 Cer) and d 7 -S-1-P (both Avanti Polar Lipids, Alabaster, USA) as internal standards.

Techniques: Isolation, Expressing, Quantitative RT-PCR, Mass Spectrometry, Flow Cytometry, Cell Culture