Journal: PLoS ONE

Article Title: Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties

doi: 10.1371/journal.pone.0121184

Figure Lengend Snippet: EV subpopulations were isolated from BV-2 cells, and were analyzed along with the releasing cells either directly (MVs and APOs) or after coupling to latex beads (EXOs) by flow cytometry using annexin V FITC, as well as anti-CD9 FITC, anti-CD63 PE and anti-cholesterol CF488-conjugated antibodies, and Alexa Fluor647-conjugated cholera toxin (CTX) (all marked with thick black lines), and were compared to respective negative controls (thin black lines). Isotype controls were used for samples stained with fluorochrome-conjugated antibodies, whereas autofluorescence was detected in the absence of either annexin V or CTX. Images and figures are representatives of at least three independent experiments.

Article Snippet: Annexin V-FITC (in annexin binding buffer from BD Biosciences), anti-CD9 FITC and anti-CD63 PE-conjugated antibodies (all from BD Biosciences) were added to cells and EV preparations, and were incubated for 30 min at room temperature in the dark.

Techniques: Isolation, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis

doi: 10.1101/2023.01.10.523252

Figure Lengend Snippet: Primary neurospheres obtained from the hippocampus ( A ) and the walls of the lateral ventricles ( B ) of wild-type (N=7, A; N=12, B), Kidins220 f/f (N=6, A; N=12, B), and Kidins220 Gfap Δ/Δ (N=10, A; N=15, B), mice. C , Cumulative number of cells obtained over 3 consecutive passages of SEZ neurospheres (N=7 independent cultures per genotype) and D , mean neurosphere diameter of SEZ neurospheres of wild-type (N=9), Kidins220 f/f (N=7) and Kidins220 Gfap Δ/Δ (N=8) mice. E , Representative confocal micrographs of wild-type and Kidins220 Gfap Δ/Δ neurospheres stained for the cell cycle marker Ki67 (red) and DAPI (blue) to counterstain the nuclei. Scale bar: 10 μm. F , Percentage of Ki67 + cells in neurospheres of the three genotypes (wild-type, N=3; Kidins220 f/f , N=2; Kidins220 Gfap Δ/Δ , N=3). G , Representative FACS dot-plots of NSC double-stained for Annexin V/7-AAD and H , quantitative analysis of the percentage of live cells (Annexin V - /7AAD - ) and I , of early-stage apoptotic cells (Annexin V + /7AAD - ) in wild-type (N=14), Kidins220 f/f (N=6) and Kidins220 Gfap Δ/Δ (N=11) NSCs. Data represent mean ± s.e.m. Each data point represents values from a neurosphere culture established from an individual mouse. Ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA (A, B, D, F, H, I), and two-way RM ANOVA (C), all followed by Dunnett’s post-hoc tests. 7AAD, 7-aminoactinomycin D; FACS, fluorescence-activated cell sorting.

Article Snippet: Cells were harvested by centrifugation, disaggregated, and resuspended in Annexin V Binding Buffer, PE Annexin V and 7-AAD (BD Iberia, Madrid, Spain) and analyzed in a FACSCanto-A flow cytometer (BD) using FACSDiva 6.1.3 software (BD).

Techniques: Staining, Marker, Fluorescence, FACS

Journal: bioRxiv

Article Title: Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis

doi: 10.1101/2023.01.10.523252

Figure Lengend Snippet: A , Representative immunoblots for Kidins220, p-AKT S473 , p-GSK3α/β S21/9 and GAPDH in lysates from Kidins220 Gfap Δ/Δ , Kidins220 f/f and wild-type neurospheres. Levels of p-AKT S473 (wild-type, N=6; Kidins220 f/f , N=10; Kidins220 Gfap Δ/Δ , N=3) ( B ) and p-GSK3α/β S21/9 (wild-type, N=8; Kidins220 f/f , N=10; Kidins220 Gfap Δ/Δ , N=8) ( C ) represented in arbitrary units after normalization with GAPDH, and relative to wild-type in extracts from the indicated genotypes. D , Representative immunoblots for p-GSK3α/β S21/9 and GAPDH in hippocampal lysates from wild-type, Kidins220 f/f and Kidins220 Gfap Δ/Δ mice. E , Levels of p-GSK3α/β S21/9 represented in arbitrary units after normalization with GAPDH and relative to wild-type in extracts from the indicated genotypes (wild-type, N=8; Kidins220 f/f , N=5; Kidins220 Gfap Δ/Δ , N=7). F , Numbers of primary neurospheres obtained from wild-type and Kidins220 Gfap Δ/Δ mice under low (10 ng/ml) or high (20 ng/ml) EGF mitogenic stimulation (N=3, for each condition). G , Representative phase contrast micrographs of wild-type and Kidins220 Gfap Δ/Δ primary neurospheres grown in culture medium with EGF 10 or 20 ng/ml. Neurosphere borders are outlined in orange for clarity. Scale bar 100 μm. H , Quantification of the diameters of Kidins220 Gfap Δ/Δ neurospheres grown under low and high EGF (N=3, for each condition). I , Representative immunoblots for p-GSK3α/β S21/9 and GAPDH in lysates from Kidins220 Gfap Δ/Δ and wild-type primary neurospheres grown in 20 ng/ml EGF. J , Levels of p-GSK3α/β S21/9 represented in arbitrary units after normalization with GAPDH and relative to wild-type in extracts from the indicated genotypes (N=6, for each condition). K , Quantification of the percentage of apoptotic cells in 7-AAD and Annexin V staining detected by flow-cytometry. Wild-type and Kidins220 Gfap Δ/Δ neurospheres were grown in 10 or 20 ng/ml EGF and treated with 100 nM SB216763 GSK3 inhibitor or vehicle (N=8, 5: wild-type, 10 ng/ml EGF, vehicle or SB216763, respectively; N=6, 4: Kidins220 Gfap Δ/Δ , 10 ng/ml EGF, vehicle or SB216763, respectively; N=3: Kidins220 Gfap Δ/Δ , 20 ng/ml EGF). Data represent mean ± s.e.m. Each data point represents values from a neurosphere culture established from an individual mouse (B, C), or brain extracts from an individual mouse (E). Ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.0001, by one-way ANOVA followed by Dunnett’s (B, C, E) and Bonferroni’s (F) post-hoc tests, two-tailed paired (H, K) and unpaired t -tests (J, K).

Article Snippet: Cells were harvested by centrifugation, disaggregated, and resuspended in Annexin V Binding Buffer, PE Annexin V and 7-AAD (BD Iberia, Madrid, Spain) and analyzed in a FACSCanto-A flow cytometer (BD) using FACSDiva 6.1.3 software (BD).

Techniques: Western Blot, Staining, Flow Cytometry, Two Tailed Test