mouse testicular leydig cells  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: The increase in the size of the TM3 cells due to nsPEF treatment. S: sham-exposed cells. E: nsPEF-exposed cells. ( a ) Cell diameter distribution within the samples S and E in three independent experiments, with a total of 450 cells scored for each type of sample. The data were collected 5 min after the treatment. ( b ) Cell diameter distribution within the samples S and E in one independent experiment, with 150 cells scored for each sample. The data were collected 240 min after the treatment. ( a , b ) The solid line shows the Gaussian regression for the given data. ( c , d ) The cells’ diameter in samples S and E displayed as box plots (a median value, box: 25th and 75th percentiles (bottom and top of the boxes). Whiskers: 5th and 95th percentiles) together with the corresponding images of cells recorded using the inverted optical microscope. The data corresponds to plots ( a , b ), respectively. ( e ) The fold change of forward scatter (FSC) (relative size) of nsPEF-exposed cells after the Annexin V-Alexa Fluor 488 and propidium iodide staining compared to the sham-exposed cells stained as above. The FSC signals were recorded at 0, 30, 60, and 240 min after nsPEF exposure with flow cytometry. The independent experiments were performed five (at 0 min), three (at 30 min), and two (at 60 and 240 min) times. Standard deviations for the values in the graph have been presented separately to ensure data legibility. The asterisk represents the significant difference: * p < 0.0001 in the Kolmogorov–Smirnov test, and ** p < 0.001 in the Student’s t -test, between the sham and exposed samples.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Microscopy, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: Cell morphology. SEM images. The cells were filtered through the polymer membrane and fixed with paraformaldehyde immediately after nsPEF exposure. ( a , c ) Sham samples. Morphology of TM3 cells in control samples with a folded cell membrane and numerous microvilli on the surface. ( b , d , e ) nsPEF-exposed cells. An increase in the diameter of cells, loss of microvilli, areas of roughness, and plasma membrane blebbing can be observed.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Polymer, Membrane, Control, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: Adhesion and actin cytoskeleton of TM3 cells. S: sham-exposed cells. E: nsPEF-exposed cells. ( a , b ) Wash assay: TM3 cells were stained with calcein-AM before nsPEF exposure, seeded on the plastic surface, and visualized by epifluorescence microscopy before and after washes. The wash assay was carried out at 40 ( a ) and 60 min ( b ) after nsPEF exposure. Healthy intact cells were observed without the exposure (S samples, left panels), and two populations were visible after nsPEF exposure (E samples, right panels): cells with high calcein fluorescence intensity that attached to the surface, and cells with a diminished calcein staining that were mostly washed out. The yellow bar represents 100 μm. ( c ) Differences in cell attachment between sham- and nsPEF-exposed samples were detected in the wash assay at 40 min and 60 min after treatment. The bars represent the percentage of cells that adhered to the surface (mean value ± SD of three biological replicates, each in three technical repetitions). Asterisk represents the significant difference between the fractions of cells adhering to the surface in sham- and nsPEF-exposed samples at 40 and 60 min (* p < 0.001, Student’s t -test). ( d ) Actin cytoskeleton stained with phalloidin-Alexa Fluor 488 (green), and the nucleus stained with Hoechst (blue). The cells were fixed after 10 min (upper row), and 240 min (lower row), post-treatment, and visualized under CLSM. The white bar represents 10 µm.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Staining, Epifluorescence Microscopy, Fluorescence, Cell Attachment Assay

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: The presence of phosphatidylserine and other apoptosis-related changes in TM3 cells. S: sham-exposed cells. E: nsPEF-exposed cells. ( a ) The presence of phosphatidylserine on the membrane’s outer leaflet of cells stained with AnnexinV-Alexa Fluor 488 (AnV) and propidium iodide (PI) before the treatment with nsPEF, and immediately after treatment. Four populations of TM3 cells were distinguished by flow cytometry, and 10,000 events were recorded for each sample. The results are expressed as fold changes relative to sham-exposed samples. The bars represent median value and the 75th percentile of five (cells stained before exposure), or four (cells stained after exposure), independent experiments, with three biological repetitions per condition; the difference was significant (Student’s t -test (* p < 0.05)) for AnV + /PI - cells. ( b ) The phosphatidylserine’s externalization at the surface of cells stained before the nsPEF treatment, as described above; the percentage of cell populations in the samples S and E during 60 min post-exposure. The mean value of five (at 0 min), three (at 30 min), or two (at 60 min) independent experiments, with three biological repetitions per condition. ( c ) TEM images of sham- and nsPEF-exposed TM3 cells. Healthy cells with intact plasma and nuclear membranes predominate in sham-exposed control samples (left column). Numerous cells with morphological signs of apoptosis, such as nuclear membrane damage, chromatin condensation, and irregular cell outline, can be observed after nsPEF treatment (right column). The black bar represents 5 and 2 µm, respectively.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Staining, Flow Cytometry, Clinical Proteomics, Control, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: Redox balance, metabolic activity, and reactive oxygen species production in TM3 cells. S: sham-exposed cells. E: nsPEF-exposed cells. ( a ) The mitochondrial membrane potential was assayed with a JC-1 probe and recorded with flow cytometry. C1: positive control with valinomycin. C2: positive control with CCCP. Each bar shows the mean (±SD) of four independent experiments, with at least two biological repetitions. ( b ) TM3 metabolic activity measured with a PrestoBlue ® assay. The boxes show the median value of four independent experiments, with at least three biological and six technical repetitions per condition for each experiment ( n = 68). The 25th and 75th percentiles are indicated by the bottoms and tops of the boxes, respectively. Whiskers: 5th and 95th percentiles. ( c ) Left: Formation of reactive oxygen species determined with the CellROX™ Orange Reagent, presented as a percentage of the average fluorescence values for S samples (±SD). Right: Images of cells under CLSM, the orange fluorescent signals are specific for reactive oxygen species detection, blue signals are from Hoechst-stained cells’ nuclei. ( d ) Left: Superoxide production by mitochondria measured with the MitoSOX™ Red Mitochondrial Superoxide, presented as a percentage of the average fluorescence values for S samples (±SD). Right: The arrangement of oxidized MitoSOX™ Red in the cells (red) and nuclei stained with Hoechst (blue). Asterisks represent the significant difference (* p < 0.005; the Student’s t -test) between the samples S and E. The white bar represents 10 µm.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Activity Assay, Membrane, Flow Cytometry, Positive Control, Prestoblue Assay, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: Synthesis and damage of DNA in TM3 cells. S: sham-exposed cells. E: nsPEF-exposed cells. ( a – d ) The extent of DNA damage was evaluated 30 min after exposure by the neutral comet assay and expressed as ( a ) tail DNA or ( b ) tail moment. C1: the cells treated with 350 µM etoposide for 24 h, positive control. Each box shows the median value (the horizontal line within a box) of four independent experiments. The 25th and 75th percentiles are indicated by the bottom and top of the boxes, respectively. Whiskers: 5th and 95th percentiles. A significant difference between sham- and nsPEF-exposed cells was determined with the nonparametric Mann–Whitney U test (* p < 0.001). ( c ) The percentages of cells in comet stage categories. Each bar shows the mean (±SD) of four independent experiments. Asterisks represent the significance level (* p < 0.001, ** p < 0.05, Student’s t-test) of the difference between the sham and exposed samples. ( d ) Photomicrographs of four comet stages (A–D) corresponding to nuclear DNA content in the tail observed under an epifluorescence microscope with 20 × objective lens magnification. ( e ) Synthesis of DNA by TM3 cells assessed with the CyQuant ® assay 24 h after exposure. The boxes show the median value of four independent experiments, with three biological replicates per condition. The 25th and 75th percentiles are indicated by the bottom and top of the boxes, respectively. Whiskers: 5th and 95th percentiles. ( f – g ) The cell cycle phases at 4 h (f) and 24 h. ( g ) After sham and nsPEF exposure of TM3 cells. Asterisks represent the significance level (*** p < 0.00001, **** p < 0.005, chi-square test) of the differences between the sham and exposed samples.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Neutral Comet Assay, Positive Control, MANN-WHITNEY, Microscopy, CyQUANT Assay

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: Transcriptome analysis of TM3 cells. S: sham-exposed cells. E: nsPEF-exposed cells. ( a ) Venn diagram for groups of differentially expressed genes identified by a two-way ANOVA with interaction (independent variables: treatment and time; p < 0.05). Orange circle: treatment-dependent differentially expressed genes. Red circle: time-dependent differentially expressed genes. Blue circle: treatment- and time-dependent differentially expressed genes. ( b ) Upper panel: unsupervised hierarchical clustering performed for all differentially expressed genes identified by ANOVA. Red colour indicates a relatively higher gene expression, and the blue, a relatively lower gene expression. Clustering on genes and conditions. Similarity measure: Euclidean. Linkage rule: Ward’s. Four main gene clusters are indicated in different colours. Lower panel: close-up of cluster 2 showing five subclusters of genes with a higher expression in nsPEF-exposed samples.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells

doi: 10.3390/ijms222011236

Figure Lengend Snippet: Gene set enrichment analysis for TM3 cells. E0: nsPEF-exposed cells, time 0 h; E4: nsPEF-exposed cells, time 4 h; E24: nsPEF-exposed cells, time 24 h. The most negatively enriched hallmark gene sets identified in the gene set enrichment analysis comparing nsPEF-exposed and sham samples at 0 h (left), 4 h (centre), and 24 h (right) time points. Normalised enrichment scores (NES) and false discovery rate (FDR) q-values are shown below the plots. ( a ) Hallmark: oxidative phosphorylation; genes encoding proteins involved in oxidative phosphorylation. ( b ) Hallmark: DNA repair; genes involved in DNA repair. ( c ) Hallmark: E2F targets; genes encoding cell-cycle-related targets of E2F transcription factors.

Article Snippet: Leydig TM3 mouse testicular cells (ATCC CRL-1714, Manassas, Virginia, USA) were propagated in a composed medium, DMEM/F12 (Dulbecco’s modified Eagle’s and Ham’s F12 medium, 1:1, Lonza, Basel, Switzerland), with supplements (Lonza, Basel, Switzerland): 5% horse serum, 2.5% fetal bovine serum, and antibiotic/antimycotic in 5% CO 2 at 37 °C until semiconfluent.

Techniques: Phospho-proteomics