Journal: Frontiers in Chemistry

Article Title: Recent Progress in FD-LC-MS/MS Proteomics Method

doi: 10.3389/fchem.2021.640336

Figure Lengend Snippet: Specifications of each proteomics method.

Article Snippet: Since fluorescence detectors optimized for nano-LC are not commercially available, the fluorescence detector for this system was a customized commercial LC fluorescence detector (FP-4025, JASCO) that incorporated a capillary tube with an internal diameter of 30 μm and a length of 600 mm as the flow cell. shows the fluorescence chromatograms obtained from a 1,400 min (ca.

Techniques: Nucleic Acid Electrophoresis, Fluorescence, Mass Spectrometry, Fractionation

Journal: Frontiers in Chemistry

Article Title: Recent Progress in FD-LC-MS/MS Proteomics Method

doi: 10.3389/fchem.2021.640336

Figure Lengend Snippet: Chromatograms of the (A) fluorescence detector and (B) mass spectrometer obtained for FD standard proteins dissolved in highly concentrated buffers (6 mol/l guanidine, 10 mmol/l CHAPS, 1 mmol/l EDTA, and 0.5 mmol/l TCEP).

Article Snippet: Since fluorescence detectors optimized for nano-LC are not commercially available, the fluorescence detector for this system was a customized commercial LC fluorescence detector (FP-4025, JASCO) that incorporated a capillary tube with an internal diameter of 30 μm and a length of 600 mm as the flow cell. shows the fluorescence chromatograms obtained from a 1,400 min (ca.

Techniques: Fluorescence, Mass Spectrometry

Journal: Frontiers in Chemistry

Article Title: Recent Progress in FD-LC-MS/MS Proteomics Method

doi: 10.3389/fchem.2021.640336

Figure Lengend Snippet: Chromatograms of the (A) fluorescence detector and (B) mass spectrometer obtained for DAABD-labeled human (K562) cell–extracted proteins over 1,400 min gradient conditions.

Article Snippet: Since fluorescence detectors optimized for nano-LC are not commercially available, the fluorescence detector for this system was a customized commercial LC fluorescence detector (FP-4025, JASCO) that incorporated a capillary tube with an internal diameter of 30 μm and a length of 600 mm as the flow cell. shows the fluorescence chromatograms obtained from a 1,400 min (ca.

Techniques: Fluorescence, Mass Spectrometry, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Hippo/Mst signaling coordinates cellular quiescence with terminal maturation in iNKT cell development and fate decisions

doi: 10.1084/jem.20191157

Figure Lengend Snippet: Dynamic regulation of iNKT cell metabolism during development and the Mst1 dependence. (A and B) Basal OCR (A) and ECAR (B) in WT thymic CD4 + CD8 + (DP), S1–S2 iNKT (combined), and S3 iNKT cells. (C) OCR in WT DP, S1–S2 iNKT, and S3 iNKT cells after the addition of oligomycin (Oligo), FCCP, and rotenone (Rot). (D) SRC (= maximum OCR – baseline OCR) in WT S1–S2 iNKT and S3 iNKT cells. (E) Basal OCR/ECAR ratio in WT DP, S1–S2 iNKT, and S3 iNKT cells. (F) Confocal microscopy analysis of Tom20 (red, staining mitochondria) and actin (green) in WT DP, S1–S2 iNKT, and S3 iNKT cells (left). Scale bar, 5 µm. Right, quantification of mitochondrial volume per cell (μm 3 ; n = 10 cells/group). (G) Quantification of mitochondrial diameter (μm) using STORM imaging of WT DP, S1–S2 iNKT, and S3 iNKT cells. (H) Enriched pathways identified by GSEA from GO and HALLMARK gene sets in Mst1-deficient S3 iNKT cells compared with WT S3 iNKT cells. (I) Basal OCR and ECAR in thymic WT and Cd4 Cre Mst1 fl/fl S3 iNKT cells ( n = 3 wells/group combined from 15 mice/group). (J) MFI of MitoTracker (left) and TMRM (right) in thymic S1, S2, and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice ( n = 3 or 4 mice/group). Histogram plots represent flow cytometry analysis of MitoTracker and TMRM in thymic S3 iNKT cells. (K) Confocal microscopy analysis (left) of Tom20 (red, staining mitochondria) and actin (green) in thymic WT and Cd4 Cre Mst1 fl/fl S3 iNKT cells and quantification (right) of mitochondrial volume per cell (μm 3 ; n = 2 mice/group; n = 269–461 total cells from 10 fields). Scale bar, 5 µm. (L) STORM imaging (left) and quantification (right) of mitochondrial diameter (μm) in thymic WT and Cd4 Cre Mst1 fl/fl S3 iNKT cells ( n = 2 mice/group; n = 35–37 cells imaged/group). Scale bar, 1 µm. (M) Flow cytometry analysis (left) and MFI (right) of CellROX (cellular ROS) in thymic S1, S2, and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice ( n = 4 or 5 mice/group). (N) Flow cytometry analysis of phosphorylated S6 (pS6) in thymic S1–S2 and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice stimulated with or without PMA/ionomycin (PMA/iono) for 30 min ( n = 4–6 mice/group). Data are relative to the average pS6 MFI for WT cells. (O) MFI of ex vivo 2-NBDG uptake in S1, S2, and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice ( n = 5 mice/group). Numbers in the plots indicate MFI (J, M, and N). Data in plots indicate mean ± SEM (A–G and I–O). *, P < 0.05, **, P < 0.01, ***, P < 0.001. One-way ANOVA with Tukey’s test (A, B, and E–G), two-way ANOVA with Bonferroni’s test (C), or two-tailed unpaired Student’s t test (D, I, and J–O). Data are representative of two independent experiments (A–E, I, K, and L), or combined from two (F, G, and L) or three (J and M–O) independent experiments.

Article Snippet: For staining mitochondria, intracellular ROS, and 2-NBDG uptake, unfixed lymphocytes were incubated for 30 min at 37°C with 10 nM MitoTracker Green or MitoTracker Deep Red (Life Technologies, M7514 and MM22426), 10 nM TMRM (ImmunoChemistry Technologies, 9105), or 2.5 μM CellROX Deep Red (Life Technologies, C10422), or for 1 h at 37°C with 30 μM 2-NBDG (Thermo Fisher Scientific, N13195) before staining surface markers.

Techniques: Confocal Microscopy, Staining, Imaging, Flow Cytometry, Ex Vivo, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Hippo/Mst signaling coordinates cellular quiescence with terminal maturation in iNKT cell development and fate decisions

doi: 10.1084/jem.20191157

Figure Lengend Snippet: Opa1 enforces S3 iNKT and iNKT1 cell development and quiescence-associated features. (A) Immunoblot analysis of Opa1, VDAC (mitochondrial loading control), and β-actin (total protein loading control) in thymic S1–S2 and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice. Opa1 expression was quantified relatively to VDAC expression ( n = 3–6 samples/group). (B and C) Flow cytometry analysis (upper) and percentages (lower) of total thymic iNKT cells and developmental stages (S1, S2, and S3; B) and effector cell subsets (C) in the thymus of WT and Cd4 Cre Opa1 fl/fl mice. (D) Flow cytometry analysis (left) and quantification of relative MFI (right) of MitoTracker (upper) or TMRM (middle) and percentages of Ki67 + cells (lower) in thymic S1, S2, and S3 iNKT cells from WT and Cd4 Cre Opa1 fl/fl mice ( n = 5 mice/group). MitoTracker and TMRM expression is relative to average MFI of WT S1 iNKT cells. (E) Confocal microscopy analysis (upper) of Tom20 (red, staining mitochondria) and actin (green) in S1–S2 and S3 iNKT cells from the thymus of WT and Cd4 Cre Opa1 fl/fl mice and quantification (lower) of mitochondrial volume per cell (μm 3 ; n = 2 mice/group; n = 20–91 total cells from three fields). Numbers indicate MFI or percentage of cells in gates or quadrants (B–D). Data in plots indicate mean ± SEM (A–E). *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Two-tailed unpaired Student’s t test (A–E). Data are combined from three (A) or four (B–D) or representative of two (E) independent experiments.

Article Snippet: For staining mitochondria, intracellular ROS, and 2-NBDG uptake, unfixed lymphocytes were incubated for 30 min at 37°C with 10 nM MitoTracker Green or MitoTracker Deep Red (Life Technologies, M7514 and MM22426), 10 nM TMRM (ImmunoChemistry Technologies, 9105), or 2.5 μM CellROX Deep Red (Life Technologies, C10422), or for 1 h at 37°C with 30 μM 2-NBDG (Thermo Fisher Scientific, N13195) before staining surface markers.

Techniques: Western Blot, Control, Expressing, Flow Cytometry, Confocal Microscopy, Staining, Two Tailed Test