Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: Tacrolimus influences F-actin cytoskeletal re-arrangement in the human-derived first-trimester extravillous trophoblast cells. ( A – C ): Single cell confocal images of control ( A ) and TAC-treated HTR8/SVneo cells ( B , C ) labeled with the CellMask Green TM Actin tracking stain. F-actin is mostly distributed in the form of stress fibers running across the cell body of untreated cells (white arrows in ( A1 , A2 )). 10 min pre-incubation with TAC resulted in a global reorganization of the F-actin filaments manifested in the formation of cortical fibers (white arrows in ( B1 , B2 )). Notably, filopodia-like structures (white arrows in ( C1 , C2 )) were observed among TAC-treated HTR8/SVneo cells evidently demonstrating a tangible outcome of the influence of TAC on F-actin cytoskeletal reorganization suggestive of cell motility. Green: CellMask Green TM -labled F-actin, Blue: DAPI-stained nuclei. Scale bars: ( A – C ) 10 µm. Nuclei were counterstained with DAPI in ( A – C ).

Article Snippet: Live HTR8/SVneo cells were pre-incubated with a CellMask Green TM actin tracking stain for 30 min prior to image acquisition using the OkoLab stage-top microscope incubator (OkoLab Bold Line, Pozzuoli, Italy) live imaging system connected to a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Techniques: Derivative Assay, Control, Labeling, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: The influence of [Ca 2+ ]i-release inhibitors and chelators on the structural distribution of F-actin in TAC-treated HTR8/SVneo cells. ( A – F ): Time-dependent cytoskeletal reorganization of F-actin in the HTR8/SVneo cells in response to Ionomycin ( A1 , A2 ), TAC ( B1 , B2 ), 2-APB ( C1 , C2 ), U73122 ( D1 , D2 ), Wortmannin ( E1 , E2 ) and PABTA ( F1 , F2 ), respectively. Note the characteristic distribution of the stress fibers throughout the cytoplasm (solid white arrows) versus the peripherally condensed cortical fiber (dashed white arrows). Failure of F-actin cellular reorganization in the 2-APB-inhibited cells (( C1 ) vs. ( C2 )) indicates the dependence of TAC actions on the functional IP3R-signaling pathway. Distinctly, unlike pre-incubation with U73122 ( D1 , D2 ) and Wortmannin ( E1 , E2 ), the presence of 2-APB ( C1 , C2 ) and PABTA ( F1 , F2 ) compromised the structural integrity and consequently the visualization of the F-actin cytoskeleton in HTR8/SVneo cells. The recording after the addition of TAC to the inhibitor pre-treated cells was 6 min. Green: CellMask Green TM -labled F-actin, Blue: DAPI-stained nuclei. Scale bars: ( A1 – F2 ) 45 µm.

Article Snippet: Live HTR8/SVneo cells were pre-incubated with a CellMask Green TM actin tracking stain for 30 min prior to image acquisition using the OkoLab stage-top microscope incubator (OkoLab Bold Line, Pozzuoli, Italy) live imaging system connected to a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Techniques: Functional Assay, Incubation, Staining

Journal: Microorganisms

Article Title: Fluorescent Nanoparticle Uptake by Myzocytosis and Endocytosis in Colpodella sp. ATCC 50594

doi: 10.3390/microorganisms11081945

Figure Lengend Snippet: Confocal and DIC images of young fusiform Colpodella sp. (ATCC 50594) trophozoites ( A – C ) following uptake of 100 nm nanoparticles with CellMask actin green and P. caudatus ( D ) uptake of E. coli BioParticles. ( A ) Overlay of DIC, 100 nm nanoparticles, CellMask actin green. ( B ) Overlay of DIC, DAPI, and 100 nm nanoparticles. ( C ) Overlay of DIC, DAPI, and CellMask actin green. ( D ) Overlay of DIC, DAPI and E. coli BioParticles (red, gray arrows) in cytoplasm of P. caudatus . Red arrowheads label regular bacteria in Colpodella sp. (ATCC 50594). White arrows indicate Colpodella sp. (ATCC 50594) nucleus (N), kinetoplast (K).

Article Snippet: Smears of formalin-fixed cells were also incubated with CellMask Green actin tracking stain (ThermoFisher Scientific) for cells incubated with 100 nm fluorescent nanoparticles and E.coli BioParticles, and CellMask Deep Red actin tracking stain (ThermoFisher Scientific) for cells incubated with 40 nm nanoparticles.

Techniques: Bacteria

Journal: Microorganisms

Article Title: Fluorescent Nanoparticle Uptake by Myzocytosis and Endocytosis in Colpodella sp. ATCC 50594

doi: 10.3390/microorganisms11081945

Figure Lengend Snippet: Confocal microscopy, and DIC images of Colpodella sp. (ATCC 50594) (yellow arrow) and P. caudatus trophozoites (red arrow) in myzocytosis. Following uptake of 40 nm nanoparticles and formalin fixation, cells were stained with CellMask actin red. ( A ) Overlay of DIC- and DAPI-stained cells showing Colpodella sp. (ATCC 50594) trophozoite nucleus (white arrow) and DAPI-stained nucleus and kinetoplast of the prey in the posterior food vacuole of Colpodella sp. (ATCC 50594) (red arrowhead). ( B ) Overlay of DIC, DAPI and 40 nm beads (green). ( C ) Overlay of DIC and 40 nm beads (green). ( D ) Overlay of DIC, CellMask actin red and DAPI. ( E ) Overlay of DIC and CellMask actin red. ( F ) Distribution of 40 nm beads in the cytoplasm of predator and prey. ( G ) Distribution of CellMAsk actin red in the cytoskeleton of predator and prey and in the tubular tether between predator and prey. ( H ) Overlay of DAPI, CellMask and 40 nm beads. ( I ) Overlay of DIC, CellMask actin red and 40 nm beads. ( J ) Overlay of DIC, DAPI, CellMask actin red, and 40 nm beads. The red arrowheads show the nucleus and kinetoplast of P. caudatus in the posterior food vacuole of Colpodella sp. (ATCC 50594) ( A , B , D , H , J ). The distribution of 40 nm beads (green) in the cytoplasm of predator and prey is shown ( B , C , F ). CellMask actin red distribution in the cytoskeleton and around the tubular tether (black arrowhead) is shown ( D , E , G , I , J ). Overlay of DIC, DAPI, 40 nm beads and CellMask actin (red) ( J ).

Article Snippet: Smears of formalin-fixed cells were also incubated with CellMask Green actin tracking stain (ThermoFisher Scientific) for cells incubated with 100 nm fluorescent nanoparticles and E.coli BioParticles, and CellMask Deep Red actin tracking stain (ThermoFisher Scientific) for cells incubated with 40 nm nanoparticles.

Techniques: Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: A multicellular actin star network underpins epithelial organization and connectivity

doi: 10.1101/2024.07.26.605277

Figure Lengend Snippet: (A) Confocal analysis of myosin-IIA-KI-GFP (magenta) localization in AcSs (green). Scale bar, 5 μm. (B) Statistical analyses of the signal intensity level of myosin-IIA-GFP in nodes and cables of AcSs. Normalized myosin-IIA-GFP signal intensity in AcS nodes = 0.666±0.01 (mean±S.E.M), in AcS cables = 0.034±0.001. N = 3 experiments, n = 154 cells. Mann-Whitney test, ****p<0.0001. (C) N-SIM analysis of P-MLC2 (magenta) localization in AcSs (green). Scale bar, 5 μm. (D) Statistical analyses of the signal intensity level of P-MLC2 in nodes and cables of AcSs. P-MLC2 signal intensity in AcS nodes = 0.743±0.038 (mean±S.E.M), in cables = 0.417±0.026. N = 3 experiments, n = 98 cells. Unpaired t-test, ****p<0.0001. (E) Confocal analysis of the apico-basal distribution of myosin-IIA-GFP (green) or P-MLC2 (magenta) in organoid-derived monolayers. Scale bar, 10 μm. (F) Statistical analyses of the signal intensity level of myosin-IIA-GFP and P-MLC2 in the apical and basal domain of differentiated cells. Mean apical myosin-IIA-GFP signal intensity in differentiated compartments = 725±48 (mean±S.E.M), basal myosin-IIA-GFP = 1159±72, apical P-MLC2 = 1314±63, basal P-MLC2 = 2078±135. N = 3 experiments, n (cells in differentiated compartments) = 23 cells. Two-way ANOVA, ****p<0.0001. (G) Confocal analysis of myosin-IIA-GFP (green) and P-MLC2 (magenta) distribution in an organoid-derived monolayer. Nuclei (DNA) are stained with Hoechst33342 (blue). Crypt-like domains (c) are delimited with a dotted blue line. Scale bar, 20 μm. (H) Statistical analyses of the relative signal intensity level of myosin-IIA-GFP and P-MLC2 in differentiated and crypt-like compartments. Mean myosin-IIA-GFP signal intensity in differentiated compartments = 2049±128 (mean±S.E.M), in crypt-like compartments = 1572±163, mean P-MLC2 signal intensity in differentiated compartments = 3644±224, in crypt-like compartments = 1536±176. N = 3 experiments, n (cells in differentiated compartments) = 23 cells, n (cells in crypt-like compartments) = 19 cells. Two-way ANOVA, *p = 0.012, ****p<0.0001. (I) Confocal analysis and z-projection of actin distribution in the basal domain of control or blebbistatin-treated organoid-derived monolayers. Scale bar, left panel 20 μm, right panel 10 μm. (J) Time-lapse images of CellMask actin (green) in tdTomato (magenta) organoid-derived monolayer after 1h blebbistatin treatment and then wash-out (t = 0min). Crypt-like domains (c) are delimited with a dotted blue line. Scale bar, 10 μm. (K) Statistical analysis of the mean time of AcS re-formation after blebbistatin treatment and wash-out. Mean time (min) = 51.41±1.67 (mean±S.E.M). n = 101 cells.

Article Snippet: CellMask Actin Deep Red actin tracking stain (#A57245) was from Invitrogen (Thermo Fisher Scientific).

Techniques: MANN-WHITNEY, Derivative Assay, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: Tacrolimus influences F-actin cytoskeletal re-arrangement in the human-derived first-trimester extravillous trophoblast cells. ( A – C ): Single cell confocal images of control ( A ) and TAC-treated HTR8/SVneo cells ( B , C ) labeled with the CellMask Green TM Actin tracking stain. F-actin is mostly distributed in the form of stress fibers running across the cell body of untreated cells (white arrows in ( A1 , A2 )). 10 min pre-incubation with TAC resulted in a global reorganization of the F-actin filaments manifested in the formation of cortical fibers (white arrows in ( B1 , B2 )). Notably, filopodia-like structures (white arrows in ( C1 , C2 )) were observed among TAC-treated HTR8/SVneo cells evidently demonstrating a tangible outcome of the influence of TAC on F-actin cytoskeletal reorganization suggestive of cell motility. Green: CellMask Green TM -labled F-actin, Blue: DAPI-stained nuclei. Scale bars: ( A – C ) 10 µm. Nuclei were counterstained with DAPI in ( A – C ).

Article Snippet: To investigate the effect of TAC on the dynamic distribution of F-actin within the cells, the CellMask Green TM actin tracking stain (Cat# A57243, Invitrogen) was used to detect F-actin in live cells.

Techniques: Derivative Assay, Control, Labeling, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: An Immune-Independent Mode of Action of Tacrolimus in Promoting Human Extravillous Trophoblast Migration Involves Intracellular Calcium Release and F-Actin Cytoskeletal Reorganization

doi: 10.3390/ijms252212090

Figure Lengend Snippet: The influence of [Ca 2+ ]i-release inhibitors and chelators on the structural distribution of F-actin in TAC-treated HTR8/SVneo cells. ( A – F ): Time-dependent cytoskeletal reorganization of F-actin in the HTR8/SVneo cells in response to Ionomycin ( A1 , A2 ), TAC ( B1 , B2 ), 2-APB ( C1 , C2 ), U73122 ( D1 , D2 ), Wortmannin ( E1 , E2 ) and PABTA ( F1 , F2 ), respectively. Note the characteristic distribution of the stress fibers throughout the cytoplasm (solid white arrows) versus the peripherally condensed cortical fiber (dashed white arrows). Failure of F-actin cellular reorganization in the 2-APB-inhibited cells (( C1 ) vs. ( C2 )) indicates the dependence of TAC actions on the functional IP3R-signaling pathway. Distinctly, unlike pre-incubation with U73122 ( D1 , D2 ) and Wortmannin ( E1 , E2 ), the presence of 2-APB ( C1 , C2 ) and PABTA ( F1 , F2 ) compromised the structural integrity and consequently the visualization of the F-actin cytoskeleton in HTR8/SVneo cells. The recording after the addition of TAC to the inhibitor pre-treated cells was 6 min. Green: CellMask Green TM -labled F-actin, Blue: DAPI-stained nuclei. Scale bars: ( A1 – F2 ) 45 µm.

Article Snippet: To investigate the effect of TAC on the dynamic distribution of F-actin within the cells, the CellMask Green TM actin tracking stain (Cat# A57243, Invitrogen) was used to detect F-actin in live cells.

Techniques: Functional Assay, Incubation, Staining

Journal: bioRxiv

Article Title: Tunneling nanotubes between neuronal and microglial cells allow bi-directional transfer of α -Synuclein and mitochondria

doi: 10.1101/2022.12.13.519450

Figure Lengend Snippet: (A) Single, middle stack images of microglial cells stained for F-Actin with CellMask deep red Actin tracking stain (pseudo-coloured gray; left panels) and mitochondria with MitoTracker green FM (pseudo-coloured orange, middle panels). (B) Zoomed images from the ROIs boxed in (A). (C-D) Zoomed images from the ROIs boxed in (B). Image snapshots were acquired using Nikon Eclispe Ti2 spinning disk microscope.

Article Snippet: For F-Actin staining in , CellMask deep red Actin tracking stain (1:1000 dilution, Invitrogen) was used.

Techniques: Staining, Microscopy

Journal: bioRxiv

Article Title: Tunneling nanotubes between neuronal and microglial cells allow bi-directional transfer of α -Synuclein and mitochondria

doi: 10.1101/2022.12.13.519450

Figure Lengend Snippet: (A) Schematic representation of the co-culture strategy used to assess transfer. (B) Schematic representation of secretion control. (C-F) Single, middle stack images of neuron-microglia co-culture stained for F-Actin with CellMask deep red Actin tracking stain (pseudo-coloured gray, upper panels) and mitochondria with MitoTracker Red CMXRos (pseudo-coloured orange, middle panels). White, dashed boxes indicate the ROI for zoomed images. Images were acquired using Nikon Eclipse Ti2 spinning disk microscope. (G) proportion of healthy-M→N (WT) and unhealthy-M→N (+ α -Syn) acceptor cells positive for mitochondrial particles. N=3, n=630 healthy (WT) acceptors and n=638 unhealthy (+ α -Syn) acceptors. ****p<0.0001, unpaired Student’s t-test. (H) Fold change of difference between the two acceptor populations in receiving mitochondrial particles from microglia depict a 6.27-times increase for unhealthy neuronal acceptor population. ****p<0.0001, unpaired Student’s t-test. (I) Proportion of healthy-M→N (WT) and unhealthy-M→N (+ α -Syn) acceptor cells positive for mitochondrial particles in secretion control. N=3, n=634 healthy (WT) acceptors and n=605 unhealthy (+ α -Syn) acceptors. ns: non-significant, unpaired Student’s t-test. (J) Distribution pattern of the number of mitochondrial puncta transfer in acceptor cells. Higher value for “0” is indicative of more instances of no transfer. (K) Average number of mitochondrial particles in acceptor neuronal cells. N=3, n=181 for healthy (WT) acceptors and n=446 for unhealthy (+ α -Syn) acceptors. ****p<0.0001; unpaired Student’s t-test. (L-M) Estimation statistics plots corresponding to un-normalised transfer in (G) and (J) respectively.

Article Snippet: For F-Actin staining in , CellMask deep red Actin tracking stain (1:1000 dilution, Invitrogen) was used.

Techniques: Co-Culture Assay, Staining, Microscopy

Journal: bioRxiv

Article Title: An engineered culture vessel and flow system to improve the in vitro analysis of volatile organic compounds

doi: 10.1101/2023.08.05.552027

Figure Lengend Snippet: (a) The borosilicate glass culture vessel, termed the Biodome, containing 5mL RPMI 1640 media. T-25 gas permeable flask caps were allowed to rest over the inlet and outlet of the vessel while in an incubator. (b) Placement of the Biodome during VOC sampling shows the glass vessel is fully submerged in beads at 37°C. Also shown is the interface between the Biodome, TDT adapter, and TDT. A sterile rubber stopper and PTFE tape are used to create an airtight seal at the interface between the TDT and the adapter. (c) 3D rendering showing the major components of the gas flow system including the hydrocarbon trap, flow meter, sterile filter, tubing, Biodome, TDT adapter, and TDT sampling tube. The compressed gas cylinder is not shown. (d) Schematic showing relevant dimensions (in mm; OD) for the Biodome culture vessel. (e) Representative GC×GC chromatogram spanning 400-2200 seconds in the first dimension and 0.5-2 seconds in the second dimension, following 24 hours of sampling SK-OV-3 ovarian adenocarcinoma cells. Fluid modeling in ANSYS TM demonstrates (f) laminar flow conditions are maintained at flow rates ≤ 20 mL/min (TDT adapter insert included) and (g) gauge pressure and (h) velocity changes due to the inflowing gas are negligible at roughly half the Biodome height, limiting turbulent interactions with the surface of the cell culture media. (i) Live/dead assay images in the Biodome show viability across 0.5 – 4 days. A T-75 flask at t = 0 (24 hours post seeding) is shown for reference. (j) Independent live-dead assays were performed using NucBlue TM (Hoechst 33342) and NucGreen TM ReadyProbes TM at 24-hour intervals, with live percentages estimated from 10 independent regions of the T-75 or Biodome culture vessels. No statistically significant differences in cell viability are observed demonstrating equivalence as compared to standard culture vessels. (k) Custom imaging tray was designed in SolidWorks ® 2019 and 3D printed to allow imaging using a Leica DMi8. (l-n) Typical SK-OV-3 morphology was visualized using fluorescent imaging and a live actin tracking stain (green color), Hoechst 33342 (blue color; live stain), and NucGreen (red color; dead stain), and no differences are observed using different culture vessels and growth environments. All brightfield and fluorescent images were taken using cells at passage < 10.

Article Snippet: We further considered cell viability through morphological fluorescence staining using a live actin tracking stain (CellMask, Invitrogen).

Techniques: Sampling, Cell Culture, Live Dead Assay, Imaging, Staining