Journal: Journal of Neuroscience

Article Title: System xc Activity and Astrocytes Are Necessary for Interleukin-1 -Mediated Hypoxic Neuronal Injury

doi: 10.1523/jneurosci.2459-07.2007

Figure Lengend Snippet: Figure 2. Role of IL-1RI signaling in the potentiation of hypoxic neuronal injury by IL-1 in vitro. A, B, Mixed cortical cell cultures were treated with 1 ng/ml IL-1 for 20–24 h in the presenceorabsenceofrIL-1ra(10–1000ng/ml;A)oranti-IL-1RI(0.1–100g/ml;B),washed, andthendeprivedofoxygen(5h).Thepercentageoftotalneuronalcelldeathwasdetermined 20–24hlater.Anasteriskindicatesvaluessignificantlygreaterthanhypoxiaalone,whereas# denotes a significant diminution of the IL-1-mediated increase in injury (IL-1) as deter- mined by one-way ANOVA followed by a Student–Newman–Keuls t test. Significance was assessed at p 0.05 (n 3–9 cultures pooled from 2–3 different experiments).

Article Snippet: In experiments using recombinant mouse IL-1ra (rIL-1ra) (R & D Systems) or monoclonal anti-mouse IL-1RI antibody directed against the extracellular domain of mouse IL-1RI (MAB7711; R & D Systems), the respective solutions were prepared with IL-1 (1.5 ng/ml; 1.5 ) and added as described above.

Techniques: In Vitro

Journal: Nature Communications

Article Title: Midkine activation of CD8 + T cells establishes a neuron–immune–cancer axis responsible for low-grade glioma growth

doi: 10.1038/s41467-020-15770-3

Figure Lengend Snippet: a Activated T cell conditioned medium (act-Tm CM) treatment stimulated WT microglia (MG) to produce a higher level of Ccl5 (ELISA) compared to non-activated T cell-conditioned medium (non-act-Tm CM). Non-activated and activated T cell CM, and CM collected from microglia alone served as controls for these experiments. b WT T cells isolated from the mouse spleen were seeded at the concentration of 2.5 × 10 6 cells ml −1 in complete PRIM1640 medium followed by 2 days of CD3/CD28 stimulation (activated; act-Tm) or vehicle (PBS) treatment (non-activated; non-act-Tm). CM was collected for chemokine array triplicates. Increased levels of TNF-α, GM-CSF, Ccl2, Ccl1, Ccl3, Ccl4, Ccl5, Il-1ra, and Il-2 expression were observed in activated T cell CM relative to non-activated T cell CM. The fold increases and P values relative to control groups for all three replicates (Supplementary Fig. ) are collated in the table. c ELISA assays reveal increased levels of TNFα, GM-CSF, Ccl2, Ccl1, Ccl3, Ccl4, Ccl5, Il-1ra, and Il-2 in the CM of activated, relative to non-activated, T cells. d WT microglia were stimulated with these differentially expressed cytokines [TNF-α (400 pg ml −1 ), GM-CSF (1000 pg ml −1 ), Ccl2 (80 pg ml −1 ), Ccl1 (500 pg ml −1 ), Ccl3 (8000 pg ml −1 ), Ccl4 (6000 pg ml −1 ), Il-1ra (80 pg ml −1 ), and Il-2 (6000 pg ml −1 )] for 24 h at the concentrations detected in the activated T cell CM. Ccl5 production by microglia was increased following Ccl4 (6000 pg ml −1 ) treatment. Veh: vehicle. e Ccl5 ELISA revealed that activated T cell CM induction of microglial Ccl5 production was reduced following treatment with increasing concentrations of Ccl4 neutralizing antibody. f Microglial Ccr5 and Ccr8 expression was validated using spleen as a positive control. g Increasing concentrations of maraviroc (MCV, Ccr5 receptor inhibitor) and AZ084 (Ccr8 receptor inhibitor) reduced T cell induction of microglial Ccl5 expression. The combination of MCV and AZ084 exhibited the greatest inhibition of microglial Ccl5 expression. All data are presented as the mean ± SEM. a This representative experiment was conducted with n = 3 independent biological samples, and was replicated two additional times with similar results. b n = 3 independent biological samples were examined over three independent experiments, as illustrated in Fig. . c and d Bar graphs represent the means ± SEM of n = 3 independent biological samples. e This representative experiment was conducted with 0 mg ml −1 anti-Ccl 4 , n = 6; 1, 2, 2.5 mg ml −1 anti-Ccl4, n = 3, independent biological samples, and was replicated two additional times with similar results. f Bar graphs represent the means ± SEM of n = 4 independent biological samples. g This representative experiment was conducted with (from left to right) n = 7, n = 6, n = 6, n = 4, n = 5, n = 4, and n = 5 independent biological samples, and was replicated two additional times with similar results. a , d , e , g One-way ANOVA with Bonferroni post-test correction; b , c , f Two-tailed Student’s t -test. Exact P values are indicated within each panel; N.S.; not significant. From left to right in each panel: a all P < 0.001, c all P < 0.001, d N.S., N.S., N.S., N.S., P = 0.035, P < 0.001, N.S., N.S.; e P = 0.025, P < 0.001, P < 0.001; f N.S.; g P = 0.012, P < 0.001, P < 0.001, P < 0.001, P < 0.001, P < 0.001.

Article Snippet: The mouse recombinant cytokines [TNFα (Abcam, ab9740), GM-CSF (R&D Systems, 415ML101), Ccl2 (R&D Systems, 279-MC-010), Ccl1 (Fisher Scientific, EMCCL1),Ccl3 (Novus Biologicals, NBP2-35193), Ccl4 (R&D Systems, 451-MB-010), Ccl5 (Fisher Scientific, 478MR025), IL-1ra (Novus Biologicals, NBP2-35105), and Il-2 (R&D Systems, 402-ML-020)] were added to microglia and the Ccl5 levels quantified.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay, Expressing, Control, Positive Control, Inhibition, Two Tailed Test