Journal: Cells

Article Title: Excessive Reactive Iron Impairs Hematopoiesis by Affecting Both Immature Hematopoietic Cells and Stromal Cells

doi: 10.3390/cells8030226

Figure Lengend Snippet: FeAS affects the expression of adhesion molecules on MS5 stroma cells and alters the profile of cytokine production. ( A ) After culture with or without FeAS, MS5 were stained as indicated and analyzed by fluorescence microscopy. Total ROS were detected with Red CC-1, intracellular labile iron with Calcein-AM, and nuclei with Hoechst 33,342. ( B ) After MS5 cells were cultured with or without FeAS for 48 h, cells were stained with the indicated Abs. Representative flow cytometry histograms from three independent experiments are shown. ( C ) Conditioned media were analyzed with a cytokine array and the accompanying kit reagents and protocol. Conditioned media from FeAS-treated (10 or 50 μM) or untreated MS5 cells (CTL) was collected after 48 h. of culture. Medium supplemented with 10% FBS αMEM was used as a negative control (NC). The assay is arranged in a 14 × 10 grid (Array 3) and a 12 × 8 grid (Array 4), with the configuration shown in . Summarized densitometry data (mean ± SD) from three independent experiments is shown in the right panel (* p < 0.05). ( D ) MS5 cells were cultured in the presence or absence of FeAS for 48 h, and co-cultured with LSKs with or without FeAS for another 48 h and the number of cobblestone-like areas seen in each well were counted under light microscopy. Summarized data (mean ± SD) from two independent experiments is shown (* p < 0.05). Unt-FeAS: FeAS was present in the culture medium only when MS5 cells were co-cultured with LSK; FeAS-CTL: FeAS was present in the culture medium when MS5 cells were cultured alone but not when they were co-cultured with LSK; FeAS-FeAS: FeAS was present in the culture medium in both, when MS5 cells were cultured alone and when they were co-cultured with LSK cells; and Unt-CTL: FeAS was not present in the culture medium neither when MS5 cells were cultured alone nor when they were co-cultured with LSK.

Article Snippet: Cells stained with Calcein-AM, RedCC-1, and Hoechst 33,342 were analyzed with a Zeiss Laser Scanning microscopy system (Zeiss LSM5 PASCAL, Obercochen, Germany).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Cell Culture, Flow Cytometry, Negative Control, Light Microscopy

Journal: eLife

Article Title: Zebrafish fin regeneration involves generic and regeneration-specific osteoblast injury responses

doi: 10.7554/eLife.77614

Figure Lengend Snippet: ( A ) Experimental scheme indicating EdU intraperitoneal (IP) injections used to label cycling osteoblasts harvested at different time points for the data presented in panels B–D. ( B ) Proliferation timeline of bglap :GFP+ osteoblasts in segment –1 as determined by EdU incorporation. N (experiments)=1, n (fins)=4 (0 hpa), 10 (1 day post amputation [dpa]), 9 (2 dpa); n (rays)=14 (0, 2 dpa), 10 (1 dpa). Error bars represent 95% CI. Kruskal-Wallis test. ( C ) Proliferation of bglap :GFP+ osteoblasts in segment –2, segment –1, and segment 0 at 2 dpa. N (experiments)=1, n (fins)=9, n (rays)=14. Error bars represent 95% CI. Dunn’s test. ( D ) bglap RNAscope expression levels of EdU+ cells in segment –1 at 2 dpa. bglap -expressing osteoblasts were categorised based on RNAscope signal intensity into ‘high’ (yellow), ‘medium’ (cyan), and ‘low’ (red) using a look-up table, with ‘high’ threshold corresponding to expression levels in segment –2. N (experiments)=1, n (fins)=9, n (rays)=12, n (cells)=62. Error bars repesent SD. Scale bar, 10 µm.

Article Snippet: To analyse cell proliferation, 5-ethynyl-2′-deoxyuridine (EdU)-Click 647 kit (baseclick GmbH BCK-EdU647) was used.

Techniques: RNAscope, Expressing