nikon® eclipse te300 inverted epifluorescence microscope  (Nikon)

Journal: Biomaterials Translational

Article Title: Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

doi: 10.12336/biomatertransl.2023.02.005

Figure Lengend Snippet: Morphological assessment of the perforated micromachined fibre mesh. (A, B) First cytotoxicity test of the 500 μm micromachined PLGA mesh. (A) Light Microscopy image of the micromachined array showing good circularity. (B) Live/dead image showing excellent cell viability and the holes clearly defined. (C–F) Light microscopy images of the 200 (C), 300 (D), 400 (E), and 500 (F) μm hole arrays, which show good circularity and even distribution. (G–J) Epifluorescent microscope images of live ADSCs on the micromachined fibre mesh with 200 (G), 300 (H), 400 (I), and 500 (J) μm hole arrays, with excellent cell viability seen across all samples, and ‘bridging’ of cells across. (K–M) Light microscope images of three selected micromachined electrospun PCL fibre mesh samples, all micromachined with the same setting but with minor variances as a result of the non-uniform thickness of the PCL nanofibre mesh. Scale bars: 500 μm. PCL: polycaprolactone; PLGA: polylactic-co-glycolic acid.

Article Snippet: For live/dead staining, the samples were stained with fluorescein and propidium iodide at day 21 to assess cell growth and viability, and then viewed under an inverted epifluorescent microscope (Nikon Eclipse TE300 Epifluorescent inverted microscope, Minato city, Tokyo, Japan).

Techniques: Light Microscopy, Microscopy

Journal: Biomaterials Translational

Article Title: Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

doi: 10.12336/biomatertransl.2023.02.005

Figure Lengend Snippet: Live/dead staining of ADSCs on the alginate and collagen stacks. (A–F) Inverted epifluorescent microscope images after live/dead staining of ADSCs on the alginate stacks. (A) Slice 1 (top slice) showing some good cell viability on day 1. (B) Slice 2 (middle slice) showing very limited cell presence indicating no cell mobility through the construct on day 1. (C) Slice 3 (bottom slice) showing no cells and thus no mobility through the cross-linked alginate on day 3. (D) Slice 1 (top slice) showing good cell viability with some stain intake by the alginate on day 7. (E) Slice 2 (middle slice) showing limited cell viability and thus mobility on day 7. (F) Slice 3 (bottom slice) showing no cell viability and thus no mobility through the alginate even on day 7. (G–L) inverted epifluorescent microscope images after live/dead staining of ADSCs on the collagen stacks. (G) Slice 1 (top slice) showing good cell viability on day 1. (H) Slice 2 (middle slice) showing good cell viability and therefore cells must be able to travel through on day 1. (I) Slice 3 (bottom slice) showing good cell viability and thus demonstrating the mobility of cells through the collagen and the micromachined fibre mesh even after 1 day. (J) Slice 1 (top slice) showing good cell viability on day 4. (K) Slice 2 (middle slice) showing good cell viability and therefore cells must be able to travel through on day 4. (L) Slice 3 (bottom slice) showing good cell viability and thus demonstrating the mobility of cells through the collagen and the micromachined fibre mesh on day 4. Scale bars: 500 μm. ADSC: adipose derived stem cell.

Article Snippet: For live/dead staining, the samples were stained with fluorescein and propidium iodide at day 21 to assess cell growth and viability, and then viewed under an inverted epifluorescent microscope (Nikon Eclipse TE300 Epifluorescent inverted microscope, Minato city, Tokyo, Japan).

Techniques: Staining, Microscopy, Construct, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: The genetic Ca 2+ sensor GCaMP3 reveals multiple Ca 2+ stores differentially coupled to Ca 2+ entry in the human malaria parasite Plasmodium falciparum

doi: 10.1074/jbc.RA120.014906

Figure Lengend Snippet: The ability of Thg but not CPA to induce SOCE is confirmed at the single-cell level. Synchronized trophozoite-stage PfGCaMP3 parasites were isolated, resuspended into MOPS buffer with 2 mm CaCl2, and plated onto glass coverslips. The cell chamber was mounted on an epifluorescence microscope, and GCaMP3 images (excitation, 488 nm; emission, 510 nm; long band pass filter) were acquired at 1 Hz. Shown are representative traces of changes in [Ca2+]c in the presence of extracellular Ca2+ in response to DMSO control (A), 10 μm CPA (B), and 5 μm Thg (C). The Ca2+ ionophore ionomycin (10 μm, Iono) was added at the end of each experiment. To assess SOCE after CPA and Thg addition, the buffer was switched to Ca2+-free MOPS buffer with 100 μm EGTA immediately prior to recording. The cells were then treated with DMSO (D), 10 μm CPA (E), or 5 μm Thg (F) for 10 min prior to CaCl2 addition (2 mm). All responses were normalized to the peak signal with 10 μm ionomycin (F/FIono). The amplitude (G) and rate of [Ca2+]c rise (H) in response to CaCl2 addition was significantly greater than the vehicle control following treatment with Thg, but not CPA. The data in G and H were averaged from at least 15 cells in each experiment and are the means ± S.D. from three or more independent experiments. In G, **, p = 0.0053; ##, p = 0.0082. In H, *, p = 0.0345; #, p = 0.0483 one-way ANOVA with Bonferroni's multiple comparison test.

Article Snippet: Once plated, the coverslips were washed twice and mounted in a 37 °C temperature-controlled cell chamber with 3 ml of imaging buffer on the stage of a wide-field epifluorescence inverted microscope (Nikon Eclipse TE300).

Techniques: Isolation, Microscopy, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: The genetic Ca 2+ sensor GCaMP3 reveals multiple Ca 2+ stores differentially coupled to Ca 2+ entry in the human malaria parasite Plasmodium falciparum

doi: 10.1074/jbc.RA120.014906

Figure Lengend Snippet: Thg blocks the CPA [Ca2+]c response, but CPA does not block the Thg [Ca2+]c response in single P. falciparum trophozoites. Synchronized PfGCaMP3 parasites were isolated, resuspended into HEPES buffer with 2 mm CaCl2, and plated on glass coverslips before loading into the incubation chamber of an epifluorescence microscope to image GCaMP3. The cells were treated with sequential additions of 10 μm CPA and 5 μm Thg as indicated (A and B) to determine whether the addition of either drug interfered with the ability of the other to mobilize Ca2+. Responses were normalized to the peak ionomycin (10 μm, Iono) response (F/FIono), and the percentage of cells responding to both drugs was measured (C). The data are the means from ≥127 cells ± S.D. from two independent experiments.

Article Snippet: Once plated, the coverslips were washed twice and mounted in a 37 °C temperature-controlled cell chamber with 3 ml of imaging buffer on the stage of a wide-field epifluorescence inverted microscope (Nikon Eclipse TE300).

Techniques: Blocking Assay, Isolation, Incubation, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: The genetic Ca 2+ sensor GCaMP3 reveals multiple Ca 2+ stores differentially coupled to Ca 2+ entry in the human malaria parasite Plasmodium falciparum

doi: 10.1074/jbc.RA120.014906

Figure Lengend Snippet: Mitochondria uncoupling does not affect [Ca2+]c responses to CPA or Thg. Synchronized PfGCaMP3 parasites were isolated, resuspended into MOPS buffer with 2 mm CaCl2, plated onto glass coverslips, and loaded into the incubation chamber of an epifluorescence microscope. The cells were loaded with 5 nm TMRE and then treated with 2.5 μm CCCP plus 2.5 μg/ml oligomycin (oligo), which uncouples the mitochondria (A). TMRE fluorescence was monitored with 543-nm excitation and 580-nm emission. In B, GCaMP3 images were acquired at 1 Hz (excitation, 488 nm; emission, 510 nm), and the cells were treated sequentially with CCCP plus oligomycin, 10 μm CPA, and 5 μm Thg. GCaMP3 signals were normalized to the peak ionomycin (10 μm, Iono) response (F/FIono).

Article Snippet: Once plated, the coverslips were washed twice and mounted in a 37 °C temperature-controlled cell chamber with 3 ml of imaging buffer on the stage of a wide-field epifluorescence inverted microscope (Nikon Eclipse TE300).

Techniques: Isolation, Incubation, Microscopy, Fluorescence