eclipse ti e widefield fluorescence microscope  (Nikon)

Journal: Frontiers in Immunology

Article Title: The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses

doi: 10.3389/fimmu.2023.1223653

Figure Lengend Snippet: Tiam1 is required for neutrophil chemotaxis but limits migration under shear stress. (A) Transwell chemotaxis with fMLP. Wild type (grey symbols) and Tiam1 –/– (blue symbols) bone marrow cells were primed with 50 ng/ml GM-CSF and 20 ng/ml TNFα for 45 min at 37˚C before seeding into the upper well of a transwell chemotaxis chamber (3 μm pores) with either 1 μM fMLP or buffer (mock) in the lower chamber and incubation for 40 or 90 min, as indicated. Neutrophils recovered from the lower chamber were identified by flow cytometry. Data are mean ± SEM of 4-5 independent experiments; each dots is the mean of one experiment. Statistics are three-way ANOVA with Sidak’s multiple comparisons test. (B) Transwell chemotaxis with CXCL1. Wild type and Tiam1 –/– cells were treated as in (A) except with 30 ng/ml or 100 ng/ml CXCL1 as the chemoattractant for 40 min. Data are mean ± SEM of 3 independent experiments (2 for 30 ng/ml). Statistics are two-way ANOVA with Sidak’s multiple comparisons test. (C) Chemotaxis on ICAM1. Purified wild type and Tiam1 –/– neutrophils were primed as in (A) , plated into an ICAM1-coated ibidi chamber (µ-slide VI 0.4), and their migration in a chemoattractant gradient with 10 µM fMLP as the highest concentration (shaded wedges, fMLP), or in buffer only (white wedges, mock), was imaged for 20 min. Cells were tracked in the steepest area of the gradient. Tracks from one representative experiment are shown. Data are mean ± SEM of 9 independent experiments; each dot is the mean of one experiment. (D) Adhesion and migration on ICAM1 under shear-stress. Wild type and Tiam1 –/– neutrophils were primed as in (A) and plated into ICAM1-coated 6-channel ibidi slides. Cells were left to adhere for 15 min at 37°C before shear flow of 6 dyn was applied, and samples were live-imaged by widefield microscopy for 10 min, with image acquisition every 10 s. Cell tracks were generated and analysed for the indicated behaviours using Fiji. Representative tracks from one experiment are shown. Data are mean ± SEM of 5 independent experiments, with 6 samples per genotype in each experiment; each dot is the mean of one experiment. Statistics in (B, C) are two-way ANOVA with Sidak’s multiple comparisons test. (A–C) P-values in black denote significant differences, p-values in grey are non-significant.

Article Snippet: To image adhesion, morphology, and F-actin dynamics in live neutrophils, ibidi μ-slide 8-well glass bottom chambers were coated with ICAM1 or pRGD, and ICAM1-coated chambers were blocked in FAF-BSA as described above, and prewarmed in the chamber (37°C, 5% CO 2 ) of a Nikon Eclipse Ti-E widefield fluorescence microscope.

Techniques: Chemotaxis Assay, Migration, Shear, Incubation, Flow Cytometry, Purification, Concentration Assay, Microscopy, Generated

Journal: Frontiers in Immunology

Article Title: The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses

doi: 10.3389/fimmu.2023.1223653

Figure Lengend Snippet: Tiam1 limits neutrophil adhesion, polarity and focal adhesions, and determines F-actin dynamics. (A) Adhesion and spreading. Wild type (grey symbols) and Tiam1 –/– (blue symbols) neutrophils were prewarmed, plated onto ICAM1-coated coverslips for 10 min at 37˚C, fixed, stained with FITC-Gr1 antibody and imaged by widefield fluorescence microscopy. Representative images from one experiment are shown. Cell masks were generated. Adhesion was quantified as the number of neutrophils per fov; spreading as the surface area of each cell mask. Data are mean ± SEM of 4 independent experiments, with 27 fov per coverslip and duplicate coverslips per condition assessed in each experiment; each dot is the mean of one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons test. (B) Cell and F-actin polarity. Wild type and Tiam1 –/– neutrophils were allowed to adhere to pRGD-coated coverslips for 15 min at 37°C, fixed, permeabilised, and stained with phalloidin-Atto 655 and Hoechst 33342. Cell morphology and F-actin localisation were analysed using widefield fluorescence microscopy, with images blinded prior to analysis. Representative images from one experiment are shown. Closed arrows denote F-actin at the leading edge, open arrows F-actin at the uropod. Data are mean ± SEM of 6 independent experiments, with 35-96 cells per genotype assessed in each experiment; each dot is the mean of one experiment. Statistics for cell polarity are paired t-test, for F-actin distribution two-way ANOVA with Sidak’s multiple comparisons test. (C) F-actin dynamics. RubyLifeact (RLA tg/+ ) and Tiam1 –/– RLA tg/+ neutrophils were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min at 37˚C. RLA tg/+ neutrophils were stained with CellTracker, mixed with unstained Tiam1 –/– RLA tg/+ neutrophils, plated onto ICAM1 with 0.75 µM fMLP, and live-imaged by widefield fluorescence microscopy for 20 min from the moment they started to adhere. See also Supplementary Movie 1 . Data are mean ± SEM of 6 independent experiments, with movies of 90 RLA tg/+ and 131 Tiam1 –/– RLA tg/+ neutrophils analysed; each dot is the mean of one experiment. Statistics are paired t-test. (D) Focal complexes. Wild type and Tiam1 –/– neutrophils were allowed to adhere to pRGD for 15 min at 37°C in the presence or absence of 0.75 µM fMLP. Cells were fixed, permeabilised, stained with vinculin antibody and Hoechst 33342, imaged by widefield fluorescence microscopy, and images blinded prior to analysis. Representative images are from one experiment in the presence of fMLP; arrows denote focal complexes. Data mean ± SEM of 5 independent experiments, with 37-80 cells analysed per condition; each dot is the mean of one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons test. (A–D) P-values in black denote significant differences, p-values in grey are non-significant.

Article Snippet: To image adhesion, morphology, and F-actin dynamics in live neutrophils, ibidi μ-slide 8-well glass bottom chambers were coated with ICAM1 or pRGD, and ICAM1-coated chambers were blocked in FAF-BSA as described above, and prewarmed in the chamber (37°C, 5% CO 2 ) of a Nikon Eclipse Ti-E widefield fluorescence microscope.

Techniques: Staining, Fluorescence, Microscopy, Generated

Journal: Nature communications

Article Title: Cholesterol esters form supercooled lipid droplets whose nucleation is facilitated by triacylglycerols.

doi: 10.1038/s41467-023-36375-6

Figure Lengend Snippet: Fig. 5 | Seipin controls CE LD nucleation sites via its TG clustering capacity. A Airyscan images of cells with endogenously GFPx7-tagged seipin starved for 3 days in 5% LPDS + DGATi for overnight, washed and loaded with 100 µM cho- lesterol cyclodextrin for 90 min, stained with Autodot to visualize lipid droplets, and imaged live. Arrowheads indicate a stable contact between seipin and a lipid droplet during 4 seconds. Scale bar = 1 µm. The experiment was independently repeated three times with similar results. B Maximum intensity projections of deconvolved widefield images of seipin-sfGFP in control and seipin NE-trap cells. (Left) Confocal images of lipid droplets in control and seipin NE-trap cells. (Right) Cells were starved for 3 days in 5% LPDS + DGATi overnight and loaded with 200 µM cholesterol cyclodextrin + DGATi for 3 h, fixed and stained with DAPI and LD540. Scale bar = 10 µm. LD area overlapping with nuclei quantified as fraction of LDs area at NE. Control n = 727 and NE-trap n = 671 cells. Mean+ SD and all individual data points. ****p < 0.0001, two-tailed student’s t test. An independent similar experi- ment presented in Supplementary Figure 5 A. C Top-view representation of CE molecules (5 mol%, shown in orange) around the seipin oligomer associated with a model ER membrane. Left: At the beginning (t = 0). Middle: At the end (t = 3 μs) of the simulation period (zoomed in to show CE association with α2–α3 helices). Each protomer is shown with a different color. Right: A close-up view showing the

Article Snippet: Lipid droplets and seipin-sfGFP in control and NE-trap cells were imaged with Nikon Eclipse Ti-E inverted widefield fluorescence microscope using a 40x air objective, NA 0.75, and 1.5x zoom.

Techniques: Staining, Control, Two Tailed Test, Membrane