Journal: EBioMedicine

Article Title: Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs

doi: 10.1016/j.ebiom.2020.102698

Figure Lengend Snippet: Cell sorting data for separation of caboxyfluorescein diactetate succinimidyl ester (CFDA-SE)-labeled bone-derived mesenchymal stem cells (BMSCs) and unlabeled nucleus pulposus cells (NPCs) following 7-day co-culture using UPAL gel. (a): Two-dimensional (2D) dot plot for co-cultured cells. The P1 gate was placed around single live cells. (b): 2D dot plot showing unlabeled NPCs in the P2 gate and CFDA-SE-labeled BMSCs in the P3 gate that were sorted. FSC-A: Forward scatter-area; FSC-W: Forward scatter-width; SSC-A: Side scatter-area; CFDA-SE-A: CFDA-SE-area. Gene expression for each cell was normalized to the housekeeping gene GAPDH and plotted on a log scale (y-axis). Data were averaged from four different rabbit NPC lines. (c): HIF-1α, (d): GLUT-1, (e): Brachyury, (f): CDMP-1, (g): TGF-β, (h): IGF-1, (i): type II collagen, and (j): aggrecan. Data are represented as the means ± SE. p -values were determined by one-way ANOVA with a post hoc Tukey–Kramer test.

Article Snippet: For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550-conjugated rabbit polyclonal antibodies against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit polyclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit polyclonal antibodies against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied.

Techniques: FACS, Labeling, Derivative Assay, Co-Culture Assay, Cell Culture, Gene Expression

Journal: EBioMedicine

Article Title: Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs

doi: 10.1016/j.ebiom.2020.102698

Figure Lengend Snippet: Nucleus pulposus (NP) marker-positive cells in rabbit NPs. (a-c): Horizontal sections of rabbit intervertebral discs (IVDs) stained for HIF-1α, GLUT-1, and Brachyury at days 1, 7, and 28. Images are representative of four replicates (Intact control, Discectomy, and BMSCs + Gel, n = 4; days 1, 7, and 28). Scale bar = 50 μm. (d–f): Percentages of NP marker-positive cells relative to total cells. (g–i): Percentages of NP marker-positive cells relative to CFDA-SE-positive cells (representative of implanted BMSCs). Data are the means ± SE. p -values were determined using the paired t -test.

Article Snippet: For HIF-1α, GLUT-1, and Brachyury staining, DyLight 550-conjugated rabbit polyclonal antibodies against HIF-1α (Novus Biologicals, Centennial, CO, USA; NB100-479R, RRID: AB_1642267), PE-conjugated rabbit polyclonal antibody against GLUT-1 (LS Bio, Seattle, WA, USA; LS-A109342-100), and unconjugated rabbit polyclonal antibodies against Brachyury (LS Bio; LS-C31179-100, RRID: AB_911118) were applied.

Techniques: Marker, Staining, Control

Journal: Cell

Article Title: Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes

doi: 10.1016/j.cell.2024.02.025

Figure Lengend Snippet: (A) Experimental strategy. Oligodendrocytes (OL; n = 66 PTA, n = 20 MDA) and neurons (n = 56 PTA) were obtained from the brains of 20 neurotypical individuals (0–104 years of age) through FANS using NEUN (neurons) and SOX10 (OL) antibodies. Single genomes were amplified using PTA or MDA and non-clonal somatic SNVs (sSNVs) and indels were called using SCAN2. Mutation distributions were compared with snATAC-seq and snRNA-seq data obtained from a subset of the 20 individuals. (B) Integrated Genomics Viewer screenshots of two sSNVs identified by SCAN2. Top, an sSNV shared by two oligodendrocytes; bottom, a private sSNV in a neuron. (C) Extrapolated genome-wide sSNV and indel burdens for OLs and neurons as a function of age. SCAN2 estimates mutation burdens for each single cell individually by adjusting for sensitivity. Trend lines are mixed-effects linear regression models; outlier single cells with abnormally high or low mutation burdens, indicated by crosses, were excluded from the linear regressions (see ). (D) Distribution of OL and neuronal sSNVs and indels in annotated gene regions. Enrichment/depletion levels are calculated by comparison with a null distribution obtained by randomly shuffling mutations across the genome followed by correction for somatic mutation detection sensitivity; error bars represent bootstrapped 95% CIs (see ). Percentages give the observed mutation count divided by the expected mutation count from the null distribution in each region. (E) Percent of somatic mutations in the total mutation catalog with HIGH, MODERATE, and LOW impact on genes, as determined by SnpEff. See also , , , and and , , and .

Article Snippet: Pellets containing nuclei were resuspended in ice-cold PBS 1X supplemented with 3mM MgCl 2 , then filtered, blocked in PBS 1X supplemented with 3mM MgCl 2 and 3% Bovine Serum Albumin (blocking solution), and stained with an anti-NEUN antibody (Millipore MAB377) previously used for neuronal nuclei isolation, , anti-SOX10 antibody (Novus NBP2-59621R), and DAPI.

Techniques: Amplification, Mutagenesis, Genome Wide, Comparison

Journal: Cell

Article Title: Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes

doi: 10.1016/j.cell.2024.02.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Pellets containing nuclei were resuspended in ice-cold PBS 1X supplemented with 3mM MgCl 2 , then filtered, blocked in PBS 1X supplemented with 3mM MgCl 2 and 3% Bovine Serum Albumin (blocking solution), and stained with an anti-NEUN antibody (Millipore MAB377) previously used for neuronal nuclei isolation, , anti-SOX10 antibody (Novus NBP2-59621R), and DAPI.

Techniques: Whole Genome Amplification, Binding Assay, dsDNA Assay, Multiplex Assay, Sequencing, Software