Cellmask orange

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, Germany

CellMask Orange is a fluorescent dye used to label the plasma membranes of cells. It is designed for live-cell imaging applications.

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CellMask (107 citations) CellMask Orange Plasma Membrane Stain (57 citations) CellMask Orange plasma membrane dye (3 citations) CellMask™ Orange stain (2 citations) CellMask Orange plasma membrane stain probe (2 citations) HCS CellMask Orange (2 citations) CellMaskTM orange plasma stain (2 citations) CellMask Orange Actin Tracking Stain (2 citations)

84 protocols using cellmask orange

Multicolor Imaging of Cell Populations

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Confocal microscopy – Fluorescent images were collected on a LSM 700 confocal microscope (Zeiss, Jena, Germany), Fig. 3: Endothelial cells were stained with CellMask Orange (plasma membrane) (Life Technologies, Carlsbad, CA) and Hoechst (nucleus) (Life Technologies, Carlsbad, CA). B-cells, T-cells, and macrophages were pre-labeled with CellTrace dyes of different wavelengths (Thermo Fisher Scientific, Waltham, MA). Fig. 5: Endothelial cells were stained with CellMask Orange (plasma membrane) (Life Technologies, Carlsbad, CA). Explanted tumor and healthy lymph node lymphocytes were stained with Hoechst (nucleus) (Life Technologies, Carlsbad, CA).
Epiflourescence microscopy – Epifluorescence images were taken on a TE2000-u microscope (Nikon, Tokyo, Japan): CellMask Orange and Syto-13 (nucleus) (Life Technologies, Carlsbad, CA). Images were taken using a 10× objective (NA: .3, Nikon, Tokyo, Japan).
Spinning disk confocal microscopy - Fluorescent images were collected on a PerkinElmer UltraVIEW VoX spinning disk confocal microscope at the Petit Institute’s Optical Microscopy Core. Image processing, stitching, and three-dimensional reconstruction were done with Volocity (PerkinElmer Inc., USA).

Mannino R.G., Santiago-Miranda A.N., Pradhan P., Qiu Y., Mejias J.C., Neelapu S.S., Roy K, & Lam W.A. (2017). 3D microvascular model recapitulates the diffuse large B-cell lymphoma tumor microenvironment in vitro. Lab on a chip, 17(3), 407-414.

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Real-Time Platelet and RBC Aggregation Analysis

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Confocal Microscopy – LSM 700 (Zeiss, Jena, Germany), CellMask Orange (plasma membrane) (Life Technologies, Carlsbad, CA) and Hoechst (nucleus) (Life Technologies, Carlsbad, CA).
Epiflourescence microscopy – TE2000-u (Nikon, Tokyo, Japan): CellMask Orange and Syto-13 (nucleus) (Life Technologies, Carlsbad, CA). Images were taken using either a 4x objective (NA: .13, Nikon, Tokyo, Japan), or a 10x objective (NA: .3, Nikon, Tokyo, Japan). Figure 3h contains 3 images stitched together.
Videos of platelet and RBC aggregation were obtained via epifluorescence microscopy and processed with LabVIEW (National Instruments) frame by frame in a loop, which was executed for the entire length of the video (5 minutes). Using fluorescently labeled RBCs and platelets, a minimum threshold filter, based on fluorescence intensity, was applied to each video frame to identify and locate aggregates. Aggregation size was determined in each frame and plotted versus time.

Mannino R.G., Myers D.R., Ahn B., Wang Y., Margo R.o.l.l.i.n.s., Gole H., Lin A.S., Guldberg R.E., Giddens D.P., Timmins L.H, & Lam W.A. (2015). “Do-it-yourself in vitro vasculature that recapitulates in vivo geometries for investigating endothelial-blood cell interactions”. Scientific Reports, 5, 12401.

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Cell Wall and Septum Staining

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To stain the septum and cell wall, cells were stained in YE liquid with 50 μg/ml Calcofluor White M2R (Sigma-Aldrich, St. Louis, MO) at room temperature. Cells were washed with fresh YE liquid once before imaging. For CellMask Orange staining, cells were stained in YE liquid with 5 μg /ml CellMask Orange in dimethyl sulfoxide (Thermo Fisher Scientific) for 5 min at room temperature in the dark. Cells were washed with fresh YE liquid before imaging.

Wei B., Hercyk B.S., Mattson N., Mohammadi A., Rich J., DeBruyne E., Clark M.M, & Das M. (2016). Unique spatiotemporal activation pattern of Cdc42 by Gef1 and Scd1 promotes different events during cytokinesis. Molecular Biology of the Cell, 27(8), 1235-1245.

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Compression Imaging of Early Embryos

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For anteroposterior (AP) compression, early 4-cell stage embryo were placed in a dish containing ASW with CellMask orange (1 µg/ml; C10045, Invitrogen) with a piece of PDMS sculpted with a cavity of rectangular geometry. The compression was realized by placing a coverslip on top of the PDMS. The embryos placed in a cavity providing the proper AP compression were then imaged on an upright Leica TCS SP5 or SP8 equipped with a HC PL APO ×20/0.8 (Leica). For AV compression, early 4-cell stage embryos were placed in ASW with CellMask orange (1 µg/ml; C10045, Invitrogen) on a glass slide. Then a coverslip was gently added on the top and pressed under a stereoscope until the compression was observed. Embryos were then imaged on an inverted Leica TCS SP5 or SP8 equipped with a HC PL APO ×20/0.8 (Leica).

Godard B.G., Dumollard R., Heisenberg C.P, & McDougall A. (2021). Combined effect of cell geometry and polarity domains determines the orientation of unequal division. eLife, 10, e75639.

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Platelet Staining and Activation

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After fixation, depending on the experiment, platelets were stained with an appropriate plasma membrane dye (Cell Mask Deep Red or Cell Mask Orange, Life Technologies). In some instances, platelets were counterstained with phallodin (Alexa Fluor conjugated, Life Technologies) or phosphatidylserine with Annexin V (Alexa Fluor conjugated, Life Technologies). For detecting activated α_IIbβ₃, FITC-PAC-1 antibody (BD Biosciences) was applied to platelets after 75 minutes.

Myers D.R., Qiu Y., Fay M.E., Tennenbaum M., Chester D., Cuadrado J., Sakurai Y., Baek J., Tran R., Ciciliano J., Ahn B., Mannino R., Bunting S., Bennett C., Briones M., Fernandez-Nieves A., Smith M.L., Brown A.C., Sulchek T, & Lam W.A. (2016). Single-platelet nanomechanics measured by high-throughput cytometry. Nature materials, 16(2), 230-235.

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Confinement-based Live Imaging of ICM Cells

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E3.75 Pdgfra^H2B-GFP/+ positive embryos obtained from intercrossing of Pdgfra^H2B-GFP/+ and CD-1 or F1 hybrid were used. Isolated single ICM cells in Blast medium containing 1:10000 CellMask Orange (Life Technologies) were loaded into the BSA coated polydimethylsiloxane (PDMS) confinement devices with a fixed roof height of 8 μm, 9 μm or 10 μm. Confinement devices were designed to restrict cells between two glass plates, trapping the cells in the z-direction but allowing free movement in x- and y-direction, as first demonstrated in (Le Berre et al., 2014 (link)). To adapt the device for the use with very small cell numbers, we modified confinement channels as described in Heuzé et al. (2011) (link) by replacing the channels with pillars to create a constant, well-defined roof height. Live imaging of the cells was performed with a 6x silicon objective (UPLSAPO60XS, Olympus) on an inverted microscope (Olympus FV1200) equipped with a humidified chamber at 37°C and 5% CO₂. The bright field, GFP, and CellMask images of the cells were taken every one, two or three minutes for up to ten hours.

Yanagida A., Corujo-Simon E., Revell C.K., Sahu P., Stirparo G.G., Aspalter I.M., Winkel A.K., Peters R., De Belly H., Cassani D.A., Achouri S., Blumenfeld R., Franze K., Hannezo E., Paluch E.K., Nichols J, & Chalut K.J. (2022). Cell surface fluctuations regulate early embryonic lineage sorting. Cell, 185(5), 777-793.e20.

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Imaging Cardiac Z-disks and T-tubules

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Experiments were performed as described in our previous study (Inoue et al., 2013 (link)). In brief, the heart was isolated from the mouse anesthetized with pentobarbital sodium (100 mg/kg, intraperitoneally), and perfused via the aorta with 5 ml of Ca²⁺-free HEPES-Tyrode’s solution containing 80 mM 2,3-butane-dione monoxime (BDM) at a speed of ∼1 drop/s. Then, the heart was mounted on the custom-made microscope stage, and AcGFP-expressing Z-disks were imaged. In some experiments, CellMask Orange (5 µg/ml in the above solution, 2 ml at a speed of ∼0.5 drop/s; Life Technologies) was used to stain the T-tubules in ventricular myocytes in the isolated heart. After the treatment with CellMask Orange, the heart was perfused with 5 mL of Ca²⁺-free HEPES-Tyrode’s solution containing 80 mM BDM at a speed of ∼1 drop/s. In this case, the heart was illuminated with a 532-nm laser light (PID-1500; Snake Creek Lasers), and the resultant fluorescence signals (emission filter, BA575IF; Olympus) were detected by the EMCCD camera. A 60× lens (60×W; numerical aperture [N/A] 1.00; LUMPlanFL N; Olympus) was used. Experiments were performed at 25°C.

Kobirumaki-Shimozawa F., Oyama K., Shimozawa T., Mizuno A., Ohki T., Terui T., Minamisawa S., Ishiwata S, & Fukuda N. (2016). Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function. The Journal of General Physiology, 147(1), 53-62.

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Visualizing McTNs in Suspended Cells

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For the experiments involved in suspended free-floating cells, ibidi microfluidics chambers were coated with 1% pluronic F-127 solution for 30 minutes. Cells were treated with a 1:10,000 dilution of CellMask-Orange (Life Technologies) membrane stain in order to visualize McTNs. Next, cells were treated with the vehicle or a drug treatment of 10 μg/mL paclitaxel or 125 μM colchicine. A 150 μL sample of treated cells was added to each ibidi channel at a concentration of 30,000 cells per channel. Cells were incubated at 37C to allow absorption of CellMask and drug treatment for 30 minutes prior to imaging.

Ory E.C., Chen D., Chakrabarti K.R., Zhang P., Andorko J.I., Jewell C.M., Losert W, & Martin S.S. (2017). Extracting microtentacle dynamics of tumor cells in a non-adherent environment. Oncotarget, 8(67), 111567-111580.

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Blocking BAFF-R Signaling in B Cells

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Nilotinib and anti-BAFF-R B-1239 antibodies (unconjugated and Alexa-647 labeled) were from Novartis. These antibodies were selected from the Human Combinatorial Antibody Library (HuCAL GOLD{r}), using phage display technology (8 (link)). The B-1239 mAb was produced in a fucosyl-transferase deficient CHO cell line (BioWa Potelligent Technology, BioWa Inc., Princeton, NJ, USA) (20 (link)), with a humanized sequence to optimalize ADCC activity. B-1239 was selected based on its property of blocking the BAFF/BAFF-R interaction and signaling via BAFF. Anti-BAFF-R antibody clone 11c1, anti-human CD19 and CD10 antibodies were from BD Biosciences (San Jose, CA, USA). FITC-anti-human IgG was from Sigma Aldrich (CA, USA). Recombinant huBAFF and the function-blocking anti-BAFF-R antibodies were purchased from R&D Systems (MN, USA). Bcr N-20, BAFF-R and Gapdh antibodies for Western blotting were from Santa Cruz, eBiosciences and Millipore, respectively. Cell Mask Orange and Deep Red were from Life Technologies (Eugene, OR).

Parameswaran R., Lim M., Fei F., Abdel-Azim H., Arutyunyan A., Schiffer I., McLaughlin M.E., Gram H., Huet H., Groffen J, & Heisterkamp N. (2014). Effector-mediated eradication of precursor B acute lymphoblastic leukemia with a novel Fc engineered monoclonal antibody targeting the BAFF-R. Molecular cancer therapeutics, 13(6), 1567-1577.

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Analyzing Membrane Tethers in Breast Cancer Cells

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MDA-MB-436 cells were trypsinized, spun down, and resuspended in phenol red-free and serum-free DMEM. Cells were seeded on PMA₄/PAAm₄ coated microfluidic slides, PMA₄/PAAm₄ coated microfluidic slides with DOTAP, or a low attach 24-well plate (50,000 cells/channel). Cells were incubated for 1 hr to allow for tethering. After 1 hr, one wash was done where the existing media was gently removed from the bottom port of each channel and fresh media was added to the top port on the DOTAP slides. This wash was to ensure only tethered cells were analyzed. After this wash, CellMask Orange (Life Technologies) cell membrane dye was added to each channel to a final concentration of 1:10,000. McTNs were scored blindly in a population of 100 cells/well as previously described [12 (link)]. Representative images were taken at 40x magnification with an Olympus CKX41 fluorescence microscope.

Chakrabarti K.R., Andorko J.I., Whipple R.A., Zhang P., Sooklal E.L., Martin S.S, & Jewell C.M. (2016). Lipid tethering of breast tumor cells enables real-time imaging of free-floating cell dynamics and drug response. Oncotarget, 7(9), 10486-10497.

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