Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 1. Frequency of stem cells in DSC-fibroblast co-cultures. Dermal cells were cultured in stem cell medium for up to two weeks to form spheres. The spheres were enzymatically dissociated with Accutase™, and the percentage of DSCs (NGFRp75-positive cells) in the culture was determined via flow cytometry. (A) Boxplot displaying the median with 25th/75th percentiles. The cross (×) inside the box indicates the mean DSC frequency. Dots represent outliers. (B) The histogram shows the abundance distribution of DSC frequencies of individual donor cell strains divided into ranges of 5%. Results from 66 donors.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Cell Culture, Cytometry

Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 3. Selective detachment of DSC-fibroblast co-cultures with 10–20% stem cells. Dermal cells were incubated with Accutase™for 1, 2, or 3 min or with trypsin-EDTA for 0.5, 1, or 2 min, and detached cells were collected. (A) Frequency of DSCs in samples measured using NGFRp75 staining. (B) Viability of total cells examined with propidium iodide. (C) Plot of recovery rate (x-axis) versus purity (y-axis) of detached cells at the individual incubation times. Purity: frequency of DSCs. Recovery rate: ratio of the absolute number of DSCs in the detached sample to the absolute number of DSCs before detachment. Dotted lines indicate the DSC frequency of control cells detached with Accutase™(light gray) or trypsin-EDTA (dark gray). Results from one representative donor cell strain.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Incubation, Staining, Control

Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 5. Positive selection (labeling of DSCs). (A–C) EasySep™column-free positive selection with the EasySep™Human CD271 Positive Selection Kit II (StemCell Technologies). Sample size: n = 6. (D–F) MACS® automatic column-based positive selection with Neural Crest Stem Cell (NCSC) MicroBeads and the autoMACS® Pro Separator (Miltenyi Biotec). Program: Posseld2. Sample size: n = 13. Values are presented as mean ± SDs. Pre: initial sample, neg frac: fibroblast fraction, pos frac: DSC fraction, d11–12: 11–12 days of cultivation. Frequency of DSCs in the separate fractions was determined via flow cytometry analysis of NGFRp75 (y-axis of dot plots) and CD90 (x-axis of dot plots). Following separation, the enriched DSC fraction was cultivated in stem cell medium. After 11–12 days, the proportion of DSCs in culture was measured again via flow cytometry, and cells were additionally stained immunohistochemically for NGFRp75 (red) and CD90 (green). Nuclei were counterstained with DAPI (blue). Scale bars: 200 µm.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Selection, Labeling, Cytometry, Staining

Journal: Cells

Article Title: Enrichment of Human Dermal Stem Cells from Primary Cell Cultures through the Elimination of Fibroblasts.

doi: 10.3390/cells12060949

Figure Lengend Snippet: Figure 6. Overview of purity and recovery of DSCs after the individual selection methods. (A) Purity. Frequency of DSCs in the purified fraction after separation determined via flow cytometry analysis of NGFRp75. Values are presented as mean ± SDs. (B) Recovery rate. Ratio of the absolute number of DSCs in the purified fraction after separation to the absolute number of DSCs in the pre sample. Values are presented as mean ± SDs. (C) Plot of recovery rate (x-axis) versus purity (y-axis) of the individual selection methods. Sample size: a. n = 5; b. n = 3; c. n = 2; d. n = 3; e. n = 3; f. n = 2; g. n = 6; h. n = 13.

Article Snippet: For MACS® positive selection (labeling of DSCs), Neural Crest Stem Cell (NCSC) MicroBeads that are conjugated to anti-NGFRp75 antibodies (cat. #130-097-127; Miltenyi Biotec) were used according to the manufacturer’s protocol, with slight modifi- Cells 2023, 12, 949 6 of 20 cations: up to 1 × 107 cells were resuspended in buffer to a total volume of 160 μL (instead of 80 μL/1 × 107 cells); 40 μL of NCSC MicroBeads/1 × 107 cells (instead of 20 μL/1 × 107 cells) were added and incubated for 15 min at 4 ◦C in the dark; wash step with centrifugation at 300× g for 5 min; cell pellet was resuspended in 1 mL of buffer (instead of 500 μL), and sample was placed into the tube rack in the autoMACS® Pro Separator; the double-column positive separation program Posseld2 and the wash program Rinse were selected.

Techniques: Selection, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Interactions between Major Bioactive Polyphenols of Sugarcane Top: Effects on Human Neural Stem Cell Differentiation and Astrocytic Maturation

doi: 10.3390/ijms232315120

Figure Lengend Snippet: STEE and its major polyphenolic constituents stimulated the mitochondrial activity of hNSCs-derived immature astrocytes. ( A ) hNSCs were plated in a 96 well plate, induced differentiation into astrocytes, and pre-treated with STEE and its major constituents; 3CQA, 5CQA, 3FQA, or ISO, for the indicated times (24, 48, or 72 h). Cells were then further incubated with Rh123 for 20 min, and the fluorescence intensity generated by Rh123 was monitored at λex = 507 nm and λem = 529 nm. The percentages shown are the mean ± SEM (n = 3–6). One-way ANOVA with Dunnett’s post hoc test for the comparisons: * p < 0.05. ( B ) Cells were incubated with STEE and its constituents for 48 h, and PGC-1α mRNA ( PPARGC1 ) expression was measured. The relative values are the mean ± SEM (n = 3) compared with the control. One-way ANOVA with Dunnett’s post hoc test for the comparisons: * p < 0.05, **** p < 0.0001. A description of the number for the treatment groups is indicated in . ( C ) Phase-contrast microphotographs of hNSC-derived astrocytes cultured for 48 h in the absence or presence of STEE (50 µg/mL). High magnification views of boxed areas are shown below panels. Scale bar = 50 µm in panels and 20 μm in insets.

Article Snippet: Primary hNSCs (HS820-20f, Cell Applications, San Diego, CA, USA) were cultured as previously reported [ ].

Techniques: Activity Assay, Derivative Assay, Incubation, Fluorescence, Generated, Expressing, Control, Cell Culture

Journal: Cell reports

Article Title: A PRMT5-RNF168-SMURF2 Axis Controls H2AX Proteostasis

doi: 10.1016/j.celrep.2019.08.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GBM patient-derived cell line (#13–0302, described in Hansen et al., 2019 ) and the OMRP cell line were cultured in Neural Stem Cell medium (STEMCELL, cat# 05751) supplemented with EGF, FGF and heparin (20 ng, 10 ng, and 2 μg per ml, respectively).

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Cell Culture, Stem Cell Culture, Protease Inhibitor, Mutagenesis, CCK-8 Assay, RNA Extraction, SYBR Green Assay, Plasmid Preparation, shRNA, Software, Microscopy, Membrane