Journal: Cells

Article Title: Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis

doi: 10.3390/cells10102764

Figure Lengend Snippet: Molecular alterations on CD34 + -HSPCs and CECs. Mutated gene frequency and Molecular alterations discovered on CD34+-HSPCs ( A ) and CECs ( B ). On the top the frequency of mutated genes, while on the bottom the table with all the mutated genes in each patient.

Article Snippet: For CD34 + HSPC detection, 10 mL of PB was collected in EDTA (Ethylenediaminetetraacetic acid) tubes and examined within 6 h. HSPCs were selected using CD34+ immunomagnetic bead-column separation (magnetic-activated cell sorting (MACS) CD34 MicroBead Kit by Miltenyi biotech, 51429 Bergisch Gladbach, Germany).

Techniques:

Journal: Cells

Article Title: Comparative Mutational Profiling of Hematopoietic Progenitor Cells and Circulating Endothelial Cells (CECs) in Patients with Primary Myelofibrosis

doi: 10.3390/cells10102764

Figure Lengend Snippet: Comparative Somatic profiling of CD34 + -HSPCs and CECs in PMF patients. ( A ) Molecular profiles of both CEC and HSPC in patients with PMF. The molecular lesions found in the HSPC are in red, while in Green the ones discovered in the CEC. At the top of the table the clinical characteristics of patients, who successfully recovered CEC. ( B ) Mutated genes shared between HSPCs and CECs.

Article Snippet: For CD34 + HSPC detection, 10 mL of PB was collected in EDTA (Ethylenediaminetetraacetic acid) tubes and examined within 6 h. HSPCs were selected using CD34+ immunomagnetic bead-column separation (magnetic-activated cell sorting (MACS) CD34 MicroBead Kit by Miltenyi biotech, 51429 Bergisch Gladbach, Germany).

Techniques:

Journal: The Journal of Physiology

Article Title: Novel identification and modulation of the mechanosensitive Piezo1 channel in human myometrium

doi: 10.1113/jp283299

Figure Lengend Snippet: Figure 3. Piezo1-meidiated Ca2+ influx in CD31+ and CD31−human myometrial cells An intracellular calcium flux assay determined Piezo1 activity in phMEC (CD31+) and phUSMC (CD31−) cells. phMEC, phUSMC and HEK293 Piezo1KO cells were pre-treated with the Ca2+ indicator Calbryte (ex/em 493/515 nm) followed by exposure to Yoda1 (0.3 or 3 μM) ± the Piezo1 antagonist Dooku1 (10 μM) and the change in fluorescence (Cai2+) was measured. A, phMECs treated with 3 μM Yoda1 exhibited 4.09-fold increase in Ca2+ uptake (Cai2+) over 0.3 μM treated cells (P < 0.0001) which decreased by 35.74% when co-treated with Dooku1 (P = 0.0327) at 60 min. B, phUSMC experienced a 2.64-fold increase in fluorescence when challenged with 3 μM Yoda1 (P < 0.0001), with a respective decrease of 22.49% when co-treated with Dooku1 (P = 0.0326). C, Piezo1KO fluorescence did not vary significantly at any dose of Yoda1 or Yoda1 + Dooku1 relative to baseline (Kruskal–Wallis one-way ANOVA, P = 0.2622). D, left, ×10 bright field images of phMEC (top), phUSMC (middle), and Piezo1KO (bottom). Right, Calbryte-induced fluorescence after 3 μM Yoda1 stimulation. ×20 fluorescence images of phMEC (top), phUSMC (middle), and Piezo1KO (bottom). Data presented as ±SD.

Article Snippet: Cells were the cultured to 80% confluency, preincubated with FcR blocking reagent, then separated over CD31+ bead LS columns using a MidiMACS separator (Miltenyi Biotec,130-091-935, Auburn, CA, USA).

Techniques: Calcium Flux Assay, Activity Assay, Fluorescence

Journal: Stem Cell Reports

Article Title: A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology

doi: 10.1016/j.stemcr.2019.07.004

Figure Lengend Snippet: CM Generation from Control and Fabry iPSCs, and Their Structural Characterization (A) Cardiomyocyte differentiation and purification protocol, with FACS plots of SIRPA immunostaining (typical examples) in a differentiated population at day 11, after sphere generation, and again after column purification at day 14. (B) Levels of GL-3, normalized to phosphatidylcholine (PC) content, in control and Fabry CMs at day 28 cultured ±1 μM exogenous GL-3, and in the same populations 7 days later with the exogenous GL-3 removed (day 28 + 7). Bars represent mean ± SD from two independent experiments/differentiations. (C) Typical immunostaining of α-actinin and MLC-2V in a purified population of iPSC-CMs returned to monolayer culture. (D) Typical immunostaining of α-actinin (Z-disc), MLC-2V, and myomesin-3 (M-line) of single control and Fabry1 CMs aligned on micro-patterned glass. The schematic below highlights the sarcomeric structural organization evident from this labeling. (E) The total proportion of single cells expressing myomesin-3 or MLC-2V in an organized pattern. Note that all measured cells expressed MLC-2V. Bars represent mean ± SD from three independent experiments/differentiations (>30 cells quantified per experiment). (F) Single CM 2D area measured using fixed immunolabelled cells. The horizontal line signifies the mean. Data were collected over three independent experiments/differentiations. Scale bar, 20 μm. See also Figure S1 .

Article Snippet: CMs were purified using a MACS column-based PSC-Derived Cardiomyocyte Isolation Kit (Miltenyi, no. 130-110-188) and replated on Matrigel-coated plastic.

Techniques: Control, Purification, Immunostaining, Cell Culture, Labeling, Expressing