Celllight plasma membrane rfp

Manufactured by Thermo Fisher Scientific

CellLight Plasma Membrane-RFP is a fluorescent protein-based reagent that labels the plasma membrane of live cells. It provides a red fluorescent signal to visualize the cell surface.

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Lab products found in correlation

Mitotracker red, by Thermo Fisher Scientific (3 mentions) Bacmam 2, by Thermo Fisher Scientific (1 mentions) Fluosphere, by Thermo Fisher Scientific (1 mentions) Matlab, by MathWorks (1 mentions) Ab150113, by Abcam (1 mentions) Bodipy 493 503, by Thermo Fisher Scientific (1 mentions) Anti panck, by Abcam (1 mentions) Confocal microscopy, by Nikon (1 mentions) Ab150077, by Abcam (1 mentions) A1 confocal microscope, by Nikon (1 mentions)

4 protocols using celllight plasma membrane rfp

Assessing Endothelial Cell Proliferation and Migration

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To test for cell proliferation, CD1 periventricular endothelial cells (1 million cells per experiment) were incubated in the presence of the mitotic marker BrdU (0.05%) with or without muscimol for 1 hour to examine the impact on proliferation of these cells and processed for BrdU IHC (1 (link), 4 (link)).
In preparation for long-distance cell migration assays, square culture inserts (ibidi GmbH) were placed at one end of a 35-mm dish. Cultures of CD1-derived endothelial cells, purified from the periventricular plexus, were plated in the insert and allowed to migrate for 24 hours in endothelial cell culture medium. Endothelial cells were labeled with a cell trace marker (CellLight Plasma Membrane-RFP, BacMam 2.0, Invitrogen) to visualize endothelial cell morphologies during subsequent imaging. The migration of endothelial cells from one end of the dish to the other spanning a distance of 3.5 cm was imaged and quantified.

Subburaju S., Kaye S., Choi Y.K., Baruah J., Datta D., Ren J., Kumar A.S., Szabo G., Fukumura D., Jain R.K., Elkhal A, & Vasudevan A. (2020). NAD+-mediated rescue of prenatal forebrain angiogenesis restores postnatal behavior. Science Advances, 6(41), eabb9766.

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Contractile Forces of PASMC on Substrates

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Contractile forces exerted by PASMC on different stiffness gels were assessed by traction force microscopy essentially as described (39 (link), 46 (link), 47 (link)). Briefly, polyacrylamide substrates with shear moduli of 0.4, 1.6, and 6.4 kPa were prepared (14 (link), 45 (link)) and fluorescent sulfate–modified latex microspheres (0.2 μm, 505/515 ex/em, FluoSpheres, Invitrogen) were conjugated to the gel surfaces as described (47 (link)). PASMC were plated on fluorescent bead–conjugated discrete stiffness gels and grown for 24 hours, at which time they were treated with iloprost (10 μmol/l) or vehicle for 30 minutes before traction force measurements. Images of gel surface–conjugated fluorescent beads were acquired for each cell before and after trypsinization using a Pathway HT fluorescence imaging system (BD Biosciences) and a ×20 magnification objective. Cell area was visualized using CellLight Plasma Membrane-RFP (Invitrogen) as described (47 (link)) to delineate the cell perimeter. Tractions exerted by PASMC were estimated by measuring bead displacement fields, computing corresponding traction fields using Fourier transform traction microscopy (46 (link)), and calculating root-mean-square traction (RMST) with MATLAB (Math-Works) (46 (link)).

Liu F., Haeger C.M., Dieffenbach P.B., Sicard D., Chrobak I., Coronata A.M., Velandia M.M., Vitali S., Colas R.A., Norris P.C., Marinković A., Liu X., Ma J., Rose C.D., Lee S.J., Comhair S.A., Erzurum S.C., McDonald J.D., Serhan C.N., Walsh S.R., Tschumperlin D.J, & Fredenburgh L.E. (2016). Distal vessel stiffening is an early and pivotal mechanobiological regulator of vascular remodeling and pulmonary hypertension. JCI Insight, 1(8), e86987.

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Cell Staining and Organoid Imaging

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Cell staining with MitoTracker Red (Thermo Fisher Scientific, M22425), CellLight Plasma Membrane-RFP (Thermo Fisher Scientific, C10505), and BODIPY 493/503 (Thermo Fisher Scientific, D3922) was performed according to manufacturer’s instructions. For immunofluorescence, fixed and permeabilized cells were incubated with primary antibodies: anti-PanCK (1:200 dilution, Abcam, ab7753) plus anti-CD133 (1:200 dilution; MilliporeSigma, ZRB1013). Secondary antibodies (Abcam; ab150113 [goat anti-mouse IgG H&L, Alexa Fluor 488], ab150077 [goat anti-rabbit IgG H&L, Alexa Fluor 488]) were used at 1:1,000 dilution. Cell nuclei were counterstained with Hoechst 33342 for 15 minutes at room temperature. For PDO staining, organoids were embedded with OTC and were sectioned (5 mm), deparaffinized, and stained for histological analysis or immunofluorescence. Images were visualized with confocal microscopy (Nikon) and deconvolved with ImageJ software (NIH).

Liu Z., Lei J., Wu T., Hu W., Zheng M., Wang Y., Song J., Ruan H., Xu L., Ren T., Xu W, & Wen Z. (2023). Lipogenesis promotes mitochondrial fusion and maintains cancer stemness in human NSCLC. JCI Insight, 8(6), e158429.

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Optogenetic Regulation of Cell-Cell Adhesion

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Cells expressing either CIBN-GFP-CAAX (green) alone or CIBN-GFP-CAAX (green) and optoGEF-RhoA (red) were induced to migrate for 3 to 4 hours before imaging on a Nikon A1 confocal microscope using a 60×/1.4 NA oil-immersion objective (1024 × 128 pixels²). Cells were then visualized with the 561-nm laser to avoid triggering dimerization prematurely with the 488-nm laser. ROIs were drawn around cell-cell junctions in channels and used for targeted stimulation with a 488-nm laser set at 1% power and pulsed for 1 s at 10-s intervals for 20 intervals. ROIs were then redrawn to accommodate for cell motion, and the pulsing was repeated until leader and follower cells completely separated or until the pulsing was repeated a maximum of 20 times and no detachment had occurred. For control cells expressing only CIBN-GFP-CAAX and not optoGEF-RhoA, cells were visualized with CellLight Plasma Membrane-RFP (red fluorescent protein) (BacMam 2.0; 1:40; C10608, Thermo Fisher Scientific), which was used in accordance with the manufacturer’s specifications. These control cells were pulsed for 12 rounds of 20 pulses, which was determined to be the average number of rounds required to cause cell-cell detachment for cells that were expressing both plasmids.

Law R.A., Kiepas A., Desta H.E., Perez Ipiña E., Parlani M., Lee S.J., Yankaskas C.L., Zhao R., Mistriotis P., Wang N., Gu Z., Kalab P., Friedl P., Camley B.A, & Konstantopoulos K. (2023). Cytokinesis machinery promotes cell dissociation from collectively migrating strands in confinement. Science Advances, 9(2), eabq6480.

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