Journal: The Journal of Neuroscience

Article Title: Leading Tip Drives Soma Translocation via Forward F-Actin Flow during Neuronal Migration

doi: 10.1523/JNEUROSCI.0240-10.2010

Figure Lengend Snippet: A front-high and back-low distribution of myosin II activity exists in migrating neurons. A, B, Representative images show the distribution of phosphor-MLC (p-MLC) in a migrating (A) and a nonmigrating (B) neuron. The relative cytoplasmic level of p-MLC (green) was normalized by the staining for a cell volume marker CY3-Bis NHS Ester (red) and coded by pseudocolors (0–2). The average p-MLC signals in the whole cell of migrating (n = 11) and nonmigrating (n = 11) neurons are shown in C. Average levels of p-MLC in the GC-like tip structure (GC), shaft of the leading process (LP), and the soma of migrating and nonmigrating neurons are shown in D. Note the gradual reduction of p-MLC level from GC to soma and the significant difference between GC and soma in migrating neurons but not in nonmigrating neurons. *p < 0.05, significant difference (Student's t test). Error bars, SEM. Scale bar, 10 μm.

Article Snippet: When analyzing p-MLC signals in migrating and nonmigrating neurons, cells were costained by CY3-Bis NHS Ester (0.05 mg/ml, GE Healthcare), which labels total proteins to eliminate the cell volume effect.

Techniques: Activity Assay, Staining, Marker

Journal: Advanced Functional Materials

Article Title: A New Printable Alginate/Hyaluronic Acid/Gelatin Hydrogel Suitable for Biofabrication of In Vitro and In Vivo Metastatic Melanoma Models

doi: 10.1002/adfm.202107993

Figure Lengend Snippet: Figure 5. Visualization of the vascularization and tumor histology; a) the effect of bleaching on light microscopy (FITC-BSA perfusion in green, auto- fluorescence (AF) in red) of explanted melanoma samples (scale bar = 1 mm); b) vascularization, left: 3D reconstruction of an explant with CD31 (blue) staining with AF (green) (scale bar = 500 µm), middle: immunohistology for CD31 of the tumor (scale bar = 100 µm), right: histology PAS of the tumor (arrow indicates capillary, scale bar = 100 µm); c) environment in explants (dashed lines indicate tumor area), left: necrosis in tumor visualized via HE, middle: stromal cells positive for vimentin (VIM), macrophages positive for CD68 (scale bar = 100 µm); d) HMB-45, left: collective migration (arrow), middle and right: intravasation (arrows) (scale bar = 50 µm).

Article Snippet: Alternatively, the rats were injected intravenously with anti-CD31 antibodies (1 ml, 0.05 mg ml−1, labeled with Alexa Fluor 647, Clone TLD3A12, MCA1334A647, Bio-Rad Laboratories, Inc.).

Techniques: Light Microscopy, Fluorescence, Staining, Migration

Journal: Oncoimmunology

Article Title: Epigenetic induction of CD1d expression primes lung cancer cells for killing by invariant natural killer T cells

doi: 10.1080/2162402X.2018.1428156

Figure Lengend Snippet: iNKT cells are depleted from the blood and BAL of patients with NSCLC. PBMCs were prepared from blood samples of 9 NSCLC patients and 13 healthy donors, and macrophage-depleted cells were prepared from BAL samples from 7 NSCLC patients and 26 non-cancer subjects. Cells were stained with mAbs specific for CD3 and Vα24Jα18 and analysed by flow cytometry. A, Flow cytometric dot plot showing CD3 and Vα24Jα18 expression by PBMCs after exclusion of doublets and dead cells. B, Scatter plots showing circulating iNKT cell frequencies, as percentages of lymphocytes (left) and absolute numbers of cells per ml of blood (right) in NSCLC patients and healthy control subjects. C, Scatter plot showing iNKT cell frequencies (left) and absolute numbers (right) in BAL samples from NSCLC patients and non-cancer subjects. Statistical analysis was performed using a two-tailed t test with Welch's correction. None of the NSCLC patients studied had detectable iNKT cells in their BAL samples.

Article Snippet: Highly purified iNKT cells were expanded by culturing 1,000 iNKT cells in the wells of a 96-well round bottom microtitre plate, in iNKT cell medium (RPMI 1640 containing 0.05 mM L-glutamine, 10% HyClone FBS, 50 mg/mL streptomycin, 50 U/mL penicillin, 2.5 μg/ml amphotericin B – fungizone, 25 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids mixture and 1% essential amino acids mixture; Gibco-BRL and Thermo-Scientific) and stimulating them with the iNKT cell glycolipid ligand α-GalCer (100 ng/ml; Funakoshi; KRN7000) and 250 U/ml IL-2 (Miltenyi Biotec; 130-097-745) in the presence of an excess (2 × 10 5 ) irradiated allogeneic PBMC prepared from two donors.

Techniques: Staining, Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: Oncoimmunology

Article Title: Epigenetic induction of CD1d expression primes lung cancer cells for killing by invariant natural killer T cells

doi: 10.1080/2162402X.2018.1428156

Figure Lengend Snippet: iNKT cells degranulate in response to low-dose DAC-treated A549 cells. A549 cells were treated with PBS or DAC (50 nM, 100 nM and 1 μM) at 24, 48 and 72 hours. PBS and methanol (MeOH) were used as negative and vehicle controls. After a rest period of 3 days the cells were pulsed with vehicle, α-GalCer or 7DW8-5 for 24 hours, and then co-cultured with iNKT cells and a mAb specific for CD107a. In some wells, 10 μg/ml of a blocking antibody specific for CD1d was included. PMA and ionomycin (P/I) treatment was used as a positive control. A, Representative flow cytometry dot plot showing purity of iNKT cells as lymphocytes expressing CD3 and the Vα24Jα18 TCR. B, Flow cytometric dot plot showing CD4 and CD8 expression by gated expanded iNKT cells. C, Flow cytometric dot plot showing cell-surface CD107a expression by iNKT cells after exposure to A549 cells treated with medium (left) or α-GalCer + DAC (right). D, Mean (± SEM) frequencies of iNKT cells that expressed CD107a after co-culture with DAC-treated A549 cells pulsed with glycolipids or controls. Percentages of iNKT cells that expressed CD107a in response to PBS- or DAC-treated A549 cells were compared to those exposed to A549 cells treated with vehicle alone using a two way ANOVA with Bonferroni's multiple comparison test. Results are means of 3 experiments. E, % inhibition of cytolytic degranulation by iNKT cells in response to DAC-treated A549 cells pulsed with α-GalCer or 7DW8-5 or vehicle using an anti-CD1d mAb. Results are means of 4 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Highly purified iNKT cells were expanded by culturing 1,000 iNKT cells in the wells of a 96-well round bottom microtitre plate, in iNKT cell medium (RPMI 1640 containing 0.05 mM L-glutamine, 10% HyClone FBS, 50 mg/mL streptomycin, 50 U/mL penicillin, 2.5 μg/ml amphotericin B – fungizone, 25 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids mixture and 1% essential amino acids mixture; Gibco-BRL and Thermo-Scientific) and stimulating them with the iNKT cell glycolipid ligand α-GalCer (100 ng/ml; Funakoshi; KRN7000) and 250 U/ml IL-2 (Miltenyi Biotec; 130-097-745) in the presence of an excess (2 × 10 5 ) irradiated allogeneic PBMC prepared from two donors.

Techniques: Cell Culture, Blocking Assay, Positive Control, Flow Cytometry, Expressing, Co-Culture Assay, Comparison, Inhibition

Journal: Oncoimmunology

Article Title: Epigenetic induction of CD1d expression primes lung cancer cells for killing by invariant natural killer T cells

doi: 10.1080/2162402X.2018.1428156

Figure Lengend Snippet: iNKT cells degranulate in response to low dose DAC-treated SK-MES-1 cells. SK-MES-1 cells were treated with PBS or DAC (50 nM, 100 nM and 1 μM) at 24, 48 and 72 hours. PBS and methanol (MeOH) were used as negative and vehicle controls. After a rest period of 3 days the cells were pulsed with vehicle, α-GalCer or 7DW8-5 for 24 hours, and then co-cultured for 4 h with iNKT cells and a mAb specific for CD107a. In some wells, 10 μg/ml of a blocking antibody specific for CD1d was included. PMA and ionomycin (P/I) treatment was used as a positive control. A, Flow cytometric dot plot showing cell-surface CD107a expression by iNKT cells after exposure to medium (left) or α-GalCer + DAC (right). B, Mean (± SEM) frequencies of iNKT cells that expressed CD107a after co-culture with control SK-MES-1 or DAC-treated SK-MES-1 cells pulsed with glycolipids. Percentages of iNKT cells that expressed CD107a in response to PBS- or DAC-treated SK-MES-1 cells were compared to those exposed to SK-MES-1 cells treated with vehicle alone using a two way ANOVA with Bonferroni's multiple comparison test. Results are representative of 3 experiments. C, % inhibition of cytolytic degranulation by iNKT cells in response to DAC-treated A549 cells pulsed with α-GalCer or 7DW8-5 or vehicle using an anti-CD1d mAb. Results are means of 4 experiments. (**p < 0.01, ***p < 0.001).

Article Snippet: Highly purified iNKT cells were expanded by culturing 1,000 iNKT cells in the wells of a 96-well round bottom microtitre plate, in iNKT cell medium (RPMI 1640 containing 0.05 mM L-glutamine, 10% HyClone FBS, 50 mg/mL streptomycin, 50 U/mL penicillin, 2.5 μg/ml amphotericin B – fungizone, 25 mM HEPES, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids mixture and 1% essential amino acids mixture; Gibco-BRL and Thermo-Scientific) and stimulating them with the iNKT cell glycolipid ligand α-GalCer (100 ng/ml; Funakoshi; KRN7000) and 250 U/ml IL-2 (Miltenyi Biotec; 130-097-745) in the presence of an excess (2 × 10 5 ) irradiated allogeneic PBMC prepared from two donors.

Techniques: Cell Culture, Blocking Assay, Positive Control, Expressing, Co-Culture Assay, Control, Comparison, Inhibition