celllight tubulin-rfp  (Thermo Fisher)

Journal: Communications Biology

Article Title: Modular tissue-in-a-CUBE platform to model blood-brain barrier (BBB) and brain interaction

doi: 10.1038/s42003-024-05857-8

Figure Lengend Snippet: a The structure of the BBB was reconstructed in a CUBE using primary astrocytes and pericytes, and iPSC-derived brain microvascular endothelial cells (BMECs). (i) Differentiation of iPSC to BMEC was based on the protocol by Lippmann et al. (ii) BBB was assembled in the CUBE by first seeding astrocytes and pericytes embedded in Matrigel in the CUBE, then seeding BMECs with ROCK inhibitor Y27632 on the surface of the Matrigel after it has been cured. b The 3D structure of the BBB-in-a-CUBE was visualized on Day 6 by labelling BMECs with von Willebrand Factor (vWF), astrocytes with GFAP or CX3CR1, and pericytes with PDGFRβ or NG2, then counterstaining with DAPI. (i) was imaged at 63× magnification with 0.75 zoom factor and (ii) was imaged at 25× magnification with 1.0 zoom factor. c BBB-in-a-CUBE on Days 2, 4, and 6 from the side view was visualized by labelling astrocytes with CellLight Tubulin-RFP and pericytes with CellLight Actin-GFP, then counterstaining with DAPI. On Day 2 after seeding, the astrocytes and pericytes were evenly distributed within the Matrigel, but from Day 4 and Day 6, they can be seen to elongate towards and populate the region beneath the BMEC sheet, indicating self-organisation of the BBB structure. Scalebar = 200 µm for enlarged figure and 100 µm for all others.

Article Snippet: For experiments to visualize the movement of astrocytes and pericytes in the BBB model over the course of the culture period, astrocytes were labelled with CellLight Tubulin-RFP (Invitrogen, C10614) and pericytes with CellLight Actin-GFP (Invitrogen, C10582), overnight prior to making BBB-in-a-CUBE, according to the manufacturer’s protocol.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection

doi: 10.1101/2020.08.31.275867

Figure Lengend Snippet: (A) HepG2-CD81 cells transfected with CellLight™ rfp-α-tubulin BacMam 2.0 were infected with P. yoelii sporozoites for 6 and 24 h. Cells were fixed and stained with antibodies to UIS4. Images are maximum intensity projections from 20-25 z-slices. (B) The bar graph represents parasitophorous vacuolar membrane (PVM) and MT association. Images and the bar graph are a representative of 3 independent experiments with n ≥ 100 cells / condition. (C) HepG2-CD81 cells were infected with P. yoelii sporozoites, fixed and stained with antibodies to P. yoelii UIS4 and acetylated α-tubulin. Images are maximum intensity projections from 20-25 z-slices. (D) HepG2-CD81 cells were infected with P. yoelii sporozoites for 48 h. Nocodazole was added to the cells after 46h for 2 hours. Cells were washed and incubated for 45 sec to allow the nucleation of MT. Cells were fixed and stained with antibodies to UIS4-647 conjugate. Images and bar graph are a representative of 3 independent experiments with n ≥ 100 cells / condition. Bar = 2 μm.

Article Snippet: CellLight Tubulin-RFP / GFP, BacMam 2.0 ready transfection mix were purchased from Thermoscientific (Missouri, USA).

Techniques: Transfection, Infection, Staining, Membrane, Incubation

Journal: Biomedical Optics Express

Article Title: High-performance imaging of cell-substrate contacts using refractive index quantification microscopy

doi: 10.1364/BOE.409764

Figure Lengend Snippet: (a1-a5) and (b1-b5) show two groups of U-87 MG human neuroglioma cells. Phase contrast maps (Zeiss) were obtained using a (a1) 20× and (b2) 40× objective. (a2,a3,b3) Corresponding fluorescence maps (Leica sp8) of tubulin labeled with red fluorescent protein. (a4,b1) Corresponding bright field images (Leica Sp8). (a5) RI mosaic of three images (80μm×80μm). (b4) RI mosaic of two images (80μm×80μm). Single- and double-headed arrows indicate the lower and upper cell nuclei, respectively. The circled and squared regions in panels a5 and b4 highlight some bleb-like structures that cannot be seen in the corresponding phase contrast images in panels a1 and b2, respectively. Additionally, the squared region in panel a1 shows a fine structure that also is clearly visible in panel a5, which demonstrates the high resolution that can be achieved.

Article Snippet: For the fluorescence imaging assay, cells were transfected with CellLight Tubulin-RFP solution (Invitrogen).

Techniques: Fluorescence, Labeling