fty720 treatment  (Bio-Techne corporation)

Journal: Neuron

Article Title: Astrocyte growth is driven by the Tre1/S1pr1 phospholipid-binding G protein-coupled receptor

doi: 10.1016/j.neuron.2023.11.008

Figure Lengend Snippet: (A) Images of spinal cord astrocyte membrane labeled with myrGFP (green) and nuclei labeled with H2AmCherry (magenta) in 6 dpf Tg(slc1a3b:myrGFP-P2A-H2AmCherry) transgenic control (N=15, dorsal view; N=3 lateral view) and s1pr1vo88/vo88 mutants (N=17, dorsal view; N=5, lateral view). N, number of animals. Scale bar, 20 µm. (B) Images of sparsely labeled individual astrocytes by slc1a3b:myrGFP-P2A-H2Amcherry DNA constructs and the representative IMARIS 3D-rendering surface (grey) at 6 dpf in the spinal cord of control and s1pr1vo88/vo88 mutant zebrafish. Scale bar, 20 µm. (C) Quantification of individual astrocyte volumes in control (N=18) and s1pr1vo88/vo88 mutants (N=23) at 6 dpf, related to (B). N, number of animals. Data points represent single astrocytes. ****, p<0.0001; unpaired t test. Error bars, mean values ± S.D. (D) Images of 6 dpf Tg(slc1a3b:myrGFP-P2A-H2AmCherry) larval brain in control (N=5) and s1pr1vo88/vo88 mutants (N=6). Dashed lines mark astrocyte processes densely infiltrated neuropil in the forebrain, midbrain, and hindbrain. N, number of animals. Scale bar, 50 µm. (E) Time-lapse still images of astrocyte process dynamics labeled with myrGFP (green) in control and s1pr1vo88/vo88 mutants at 3 dpf. Dashed boxes mark the regions shown to the right. Scale bar, 20 µm. (F) Quantification of astrocyte individual process extension and retraction displacement speed in control (N=10) and s1pr1vo88/vo88 mutants (N=8) at 3 dpf. N, number of animals. Data points represent single astrocyte processes tracked. *, p<0.05; ***, p<0.001; unpaired t test. Error bars, mean values ± S.D. (G and I) Images of 6 dpf Tg(slc1a3b:myrGFP-P2A-H2AmCherry) transgenic larval spinal cord astrocytes after treatment with DMSO, 1 µM FTY720, or 1 µM Ex26 at 2–4 dpf (G) or 4–6 dpf (I). Dashed boxes represent 4 independent 10 µm x 10 µm areas in the astrocyte process-enriched regions were used to quantify the GFP coverage area percentage. Scale bar, 20 µm. (H) Quantification of relative GFP coverage area percentage at 6 dpf in the astrocyte process-enriched regions in DMSO (N=12), FTY720 (N=12), and Ex26 (N=12) after 2–4 dpf treatment. N, number of animals. Data points represent average GFP coverage area percentage of the 4 independent areas in a single fish. ****, p<0.0001; one-way ANOVA with multiple comparisons. (J) Quantification of relative GFP coverage area percentage at 6 dpf in the astrocyte process-enriched regions in DMSO (N=10), FTY720 (N=12), and Ex26 (N=11) after 4–6 dpf treatment. N, number of animals. Data points represent average GFP coverage area percentage of the 4 independent areas in a single fish. ****, p<0.0001; one-way ANOVA with multiple comparisons. See also Figure S7 and Video S2.

Article Snippet: Acquired time-lapse images were Airyscan processed, and followed by Bleach correction and 3D drift correction using Fiji (ImageJ) software before analysis. . Chemical treatments FTY720 and Ex26 (Tocris Bioscience) were dissolved in DMSO to a stock concentration of 100 mM.

Techniques: Membrane, Labeling, Transgenic Assay, Control, Construct, Mutagenesis

Journal: Mucosal Immunology

Article Title: IL-1α is required for T cell-driven weight loss after respiratory viral infection

doi: 10.1016/j.mucimm.2024.02.005

Figure Lengend Snippet: Timing of T cell recruitment to lungs affects disease timing in RSV. 6-7 weeks old BALB/c SPF mice were intranasally infected 7.7 x 10 6 PFU/ml RSV A2 subtype and culled on day 1, 3, 5, and 7 post infection. Body weight (A) and food consumed (B) were monitored daily. Immune cell populations were measured using flow cytometry (C) at each time course, and live viral plaques (D) and RSV L gene (E) were quantified. 6-7 weeks old BALB/c SPF mice were injected with 25µg of FTY720 or PBS only daily from day -2 to day 6. Mice were intranasally infected with 7.7 x 10 6 PFU/ml RSV infection on day 0. Body weight (F) and food (G) were monitored daily from day -2 to day 14. At day 7, viral load was determined using RSV L gene qPCR on RNA extracted from the left lung lobe (H) and flow cytometry was used to analyze the number of CD8 T cell (I), CD4 T cells (J) and antigen-specific CD8 + T cells (K). At day 14, viral load was determined using RSV L gene qPCR (L) and flow cytometry was used to analyze the number of CD8 T cell (M), CD4 T cells (N) and antigen-specific CD8 T cells (O). N = 5, each dot represents an individual mouse (H-O); or mean +/-SEM –A - E, F-G). Significance calculated by ordinary one-way ANOVA and post test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Experiment was repeated twice.

Article Snippet: For FTY720 treatment, FTY720 (6176, BioTechne,Minneapolis, MN, USA) was diluted in PBS to a final concentration of 25μg in 250 μl PBS.

Techniques: Infection, Flow Cytometry, Injection