Cellmask green

Manufactured by Thermo Fisher Scientific

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CellMask Green is a fluorescent dye that stains the plasma membrane of living cells. It can be used to visualize and track cell boundaries during live-cell imaging experiments.

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48 protocols using cellmask green

Labelling Actin and Plasma Membrane in Zona-Free Eggs

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F-actin was labeled with 1 μM of the live-cell Spirochrome probe SiR-actin (Cytoskeleton) which is based on natural actin-binding jasplakinolide. The plasma membrane was labeled with CellMask Green using the manufacturer’s recommended 1x working solution (ThermoFischer Scientific). Zona-free eggs were mounted on poly-L-lysine coated coverslips) with pre-warmed M2 or CaCl₂ -free M16 medium supplemented with SiR-actin, CellMask Green and incubated at 37 °C in 5% CO₂ for 30 min prior to imaging. Neither CG exocytosis or fertilization was inhibited under these experimental conditions even with 5 μm SiR-actin (Supplementary Fig. 2j, k).

Vogt E.J., Tokuhiro K., Guo M., Dale R., Yang G., Shin S.W., Movilla M.J., Shroff H, & Dean J. (2019). Anchoring cortical granules in the cortex ensures trafficking to the plasma membrane for post-fertilization exocytosis. Nature Communications, 10, 2271.

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Visualizing Cellular Organelles with Fluorescent Probes

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To highlight differences in brightness of synthetic fluorescent probes and endogenous fluorescence, we labelled cells with standard fluorescent dyes. Cells were labeled with CellMask Green (C37608, Thermo Fisher Scientific, US) in order to visualize plasma membrane. Additionally, mitochondria were stained with MitoTracker Green FM (M7514, Thermo Fisher Scientific, US). Stained cells were imaged using the spinning disk confocal microscope IXplore SpinSR (Olympus, Japan). For colocalization analysis, cells were labelled with either LysoTracker Red DND-99 (L7528, Thermo Fisher Scientific, US) or MitoTracker Red CMXRos (M7512, Thermo Fisher Scientific, US) probes and imaged using spinning disk confocal microscopy.

Uzhytchak M., Smolková B., Frtús A., Stupakov A., Lunova M., Scollo F., Hof M., Jurkiewicz P., Sullivan G.J., Dejneka A, & Lunov O. (2023). Sensitivity of endogenous autofluorescence in HeLa cells to the application of external magnetic fields. Scientific Reports, 13, 10818.

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Fluorescent Nanodiamonds for Cellular Imaging

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Dopamine hydrochloride (H8502-5G) and nocodazole (M1404-2MG) were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Tris (hydroxymethyl aminomethane) (35434-76) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Sodium azide (NaN₃) (195-11092) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Hoechst 33342 (346-07951) and the cell counting kit-8 (CCK-8) (347-07621) were purchased from Dojindo Laboratories (Kumamoto, Japan). FNDs (BR100) were purchased from FND Biotech Inc. (Taipei, Taiwan). LAMP1-mGFP plasmid, which encodes a GFP-tagged lysosome marker, was a gift from E. Dell’Angelica (Addgene plasmid no. 34831; http://n2t.net/addgene:34831; RRID: Addgene_34831). Lipofectamine 3000 Transfection Kit (3000-015) was purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) (11965-092), 0.25% trypsin-EDTA (25200-056), phosphate-buffered saline (PBS) (10010-023), penicillin and streptomycin (15140-122), fetal bovine serum (FBS) (10270-106), Alexa Fluor 488 N-hydroxysuccinimide (NHS) Ester (A20000), CellMask Green (C37608), ER-tracker (E34251), and Golgi tracker (B22650) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Sotoma S., Zhong C., Kah J.C., Yamashita H., Plakhotnik T., Harada Y, & Suzuki M. (2021). In situ measurements of intracellular thermal conductivity using heater-thermometer hybrid diamond nanosensors. Science Advances, 7(3), eabd7888.

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Flow Cytometry Validation of ABY-Decorated Microbubbles

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The DSPE-PEG-ABY conjugate amalgamated into the DPPC MBs was confirmed by flow cytometry with an anti-His-Tag-APC antibody. We added 5 µL of antibody to 100 µL of MBs (MB_B7-H3 or MB_NT) containing 1 × 10⁸ particles and the mixture was incubated at room temperature for 1 h. MBs were washed 3× in 500 µL of PBS using a microcentrifuge at 300 g for 3 min. After each wash, the upper milky layer of floating MBs was carefully separated by removing the liquid wash with a syringe needle and resuspending MBs in fresh PBS. ABY (ABY_NoHis-Tag) displayed on the surface of MB_B7-H3 was detected by flow cytometry (Guava easyCyte) of MB-bound anti-His-Tag-APC antibody and compared to MB_NT background signal or MB_B7-H3 without antibody incubation. MB-ABY fluorescence signal was also analyzed using confocal microscopy (DMi8, Leica) for both targeted and non-targeted MBs (pre-labeled with CellMask Green, ThermoFisher Scientific, Waltham, MA, USA) at 20× magnification. Acquired images were analyzed using ImageJ 1.52a software (NIH, Baltimore, MD, USA).

Bam R., Natarajan A., Tabesh F., Paulmurugan R, & Dahl J.J. (2023). Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging. International Journal of Molecular Sciences, 24(10), 9048.

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Evaluating ANO1 Localization in PSC and IRC

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H69 cholangiocytes were seeded in 8-well chambered coverglass and incubated for 6 days in H69 culture medium without fetal bovine serum that was supplemented with 20% serum of patients with PSC or IRC. Medium including patient serum was refreshed daily. Serum treatment was carried out in a blinded fashion. At day 4 after seeding, cells were transiently transfected with ANO1-mCherry. At day 6 after seeding, H69 medium was changed for Leibowitz imaging medium without phenol red (Thermo Scientific, Waltham, MS) supplemented with 20% of the respective patient serum. In addition, CellMask Green (Thermo Scientific, Waltham, MS) was added to the imaging medium to stain the plasma membrane. Microscopy was performed using a Leica TCS SP8-SMD microscope (LEICA, Wetzlar, Germany). Semiquantitative scoring of ANO1-mCherry plasma membrane staining was done separately in a blinded fashion by 3 researchers. Per ANO1-mCherry transfected cell, the ANO1-mCherry localization pattern was categorized as ‘strong membrane localization’, ‘light membrane localization’, or ‘no membrane localization’.

Herta T., Kersten R., Chang J.C., Hubers L., Go S., Tolenaars D., Paulusma C.C., Nathanson M.H., Elferink R.O., van de Graaf S.F, & Beuers U. (2021). Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection. Journal of hepatology, 76(2), 319-331.

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Multiparameter Flow Cytometry of Tissue Cells

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We closely followed the flow cytometry protocol that we previously developed for tissue suspensions.^{16 (link)} Briefly, cell suspensions were co-stained with 2.5μg/mL anti-mouse CD45-PE monoclonal antibody (clone 30-F11, BioLegend, San Diego, CA) and 0.5X CellMask Green (Thermo Fisher, Waltham, MA) for 20 minutes at 37°C. Samples were then washed twice using PBS+ by centrifugation, co-stained with 5 μg/mL 7-AAD (BD Biosciences, San Jose, CA) and 12.5 μM DRAQ5 (BioLegend) on ice for at least 15 minutes, and analyzed on an Accuri C6 Flow Cytometer (BD Biosciences). Flow cytometry data was compensated and analyzed using FlowJo software (FlowJo, Ashland, OR), and a sequential gating scheme was used to identify live and dead single tissue cells from leukocytes, red blood cells, non-cellular debris, and cellular aggregates.

Qiu X., Lombardo J.A., Westerhof T.M., Pennell M., Ng A., Alshetaiwi H., Luna B.M., Nelson E.L., Kessenbrock K., Hui E.E, & Haun J.B. (2018). Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells. Lab on a chip, 18(18), 2776-2786.

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Electroporation-Induced Cell Monolayer Changes

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Following EP, the nuclei and cell membrane of EA.hy926 cell monolayers in 24-well plates were stained with Hoechst 33258 and CellMask Green (Thermo fisher scientific, Waltham, MA both dyes at a concertation of 2 μl/ml), imaged at excitation/emission wavelengths of 460–480/500–550 nm (CellMask Green) and 340–380/420–480 nm (Hoechst 33258) at 1, 8, and 24 hours post EP with fluorescent microscopy (IX51, Olympus, Tokyo, Japan). Images were obtained at 20× magnification from at least 5 separate locations from each well and were evaluated for morphologic changes in the monolayer, resulting in loss of cell-cell contact and creation of intercellular “gaps”Image processing (ImageJ, National Institutes of Health, Bethesda, MD) was used to quantify the number, size, and distribution of such intercellular gaps. Pre- and post-EP comparison of nuclear staining were used identify gaps resulting from detached or dead cells. Confluent EA.hy926 cell monolayers in transwell inserts were treated with EP, and the transport of a small molecule dye (IR-783,MW 747 Sigma-Aldrich, St. Louis, MO) and SFB-IR783 nanoparticles was measured in the well underlying the insert at 1, 2, 4, 6, 8, 12, 24, 36, and 48 h post EP using a scanning spectrometer (Tecan, Mannedorf, Switzerland).

Kodama H., Shamay Y., Kimura Y., Shah J., Solomon S.B., Heller D, & Srimathveeravalli G. (2019). Electroporation-induced changes in tumor vasculature and microenvironment can promote the delivery and increase the efficacy of sorafenib nanoparticles. Bioelectrochemistry (Amsterdam, Netherlands), 130, 107328.

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Visualizing C. jejuni EV-induced Changes

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HeLa cells were seeded in 96-well glass bottom plates (1 × 10⁴ cells/mL; Corning) and stimulated with C. jejuni EVs (5 µg/mL) for 48 hours. After washing in PBS, cells were stained with 100 µL of 1 x CellMask Green (Thermo Fisher Scientific) and Hoechst (1:2,000; Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde (PFA) for 5 min at 37°C and washed three times with PBS. Imaging was performed with an FV1200 Confocal Microscope (Evident Australia) using a 20×, 0.75 NA objective with excitation at 405 nm for Hoechst and 488 nm for CellMask Green. Acquisition parameters were maintained at consistent settings for all images acquired. Cell cytoplasm and nucleus areas were quantified using the cell function on Imaris software (Bitplane, Oxford instruments). The algorithm used was for nucleus and cell detection. Nuclei were detected using a smooth filter width of 1.40 µm. Cell body detection used a cell smooth filter width of 1.50 µm, expanded on the nucleus, and assumed one nucleus per cell.

Le L.H., Elgamoudi B., Colon N., Cramond A., Poly F., Ying L., Korolik V, & Ferrero R.L. (2024). Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells. Microbiology Spectrum, 12(3), e03232-23.

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DNA Origami Nanostructure Fabrication

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All reagents are commercially available and used without any further purification. P8064 DNA scaffold DNA was purchased from tilibit nanosystems^® GmbH (Garching, Germany). DNA staple strands were purchased from Wuhan GeneCreate Biological Engineering Co., Ltd. (Wuhan, China). Tris(hydroxymethyl)aminomethane and EthylenediaminetetraAcetic acid disodium salt were purchased from BEIJING LIUYI BIOTECHNOLOGY CO., LTD (Beijing, China). Acetic acid and Magnesium chloride hexahydrate (MgCl₂·6H₂O) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Agarose and 5 × TBE for electrophoresis were purchased from Sangon Biotech Co., LTD (Shanghai, China). GelRed was purchased from biosharp. 1-Ethy-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and Human Serum Albumin (HSA) were purchased from Sigma-Aldrich. Hoechst33342 and Cell Mask Green were purchased from Thermo Fisher Scientific. Carbon grid was purchased from Beijing XXBR Technology Co., Ltd. (Beijing, China). Uranyl acetate solution was purchased from Beijing Zhongjingkeyi Technology Co., Ltd. (Beijing, China). Distilled water (18.2 MΩ·cm, MilliQ system) was used for all experiments.

Xu X., Fang S., Zhuang Y., Wu S., Pan Q., Li L., Wang X., Sun X., Liu B, & Wu Y. (2019). Cationic Albumin Encapsulated DNA Origami for Enhanced Cellular Transfection and Stability. Materials, 12(6), 949.

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Visualization and Quantification of Nipah Virus Fusion

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A total of 10⁴ HT-1080 cells were transfected for 4 hours with 0.1 µg each of NiV-F AU1 and NiV-G HA expression plasmids, washed, and replenished with growth media containing GRFT or 3mG. At 24 hours post transfection, cells were stained using CellMask Green (Thermo Fisher Scientific) and DAPI and viewed using a Nikon Axioscope Ti inverted fluorescence microscope at ×4 magnification. For quantitative fusion assay, 2 × 10⁴ CHO-K1 cells were transfected for 4 hours with 0.1 µg each of NiV-F AU1, NiV-G HA, and pT7-eEGFP plasmids. Negative control CHO-K1 cells were transfected with 0.2 µg of NiV-F AU1 and 0.1 µg of pT7-eGFP. Cells then were washed and replenished with growth media containing GRFT or 3mG. BSRT7/5 cells (2 × 10⁴ ) were then overlaid onto CHO-K1 cells. At ~16 hours postoverlay, green fluorescent NiV glycoprotein-induced syncytia were visualized at ×2.5 magnification on a Cytation5 instrument (BioTek) and counted from photograph montages of each well using Gen5 Software. Fluorescent objects that were both >75 µm in diameter and had a fluorescence reading >5000 were classified as positive syncytia. Additional details are found in the Supplemental Methods.

Lo M.K., Spengler J.R., Krumpe L.R., Welch S.R., Chattopadhyay A., Harmon J.R., Coleman-McCray J.D., Scholte F.E., Hotard A.L., Fuqua J.L., Rose J.K., Nichol S.T., Palmer K.E., O’Keefe B.R, & Spiropoulou C.F. (2020). Griffithsin Inhibits Nipah Virus Entry and Fusion and Can Protect Syrian Golden Hamsters From Lethal Nipah Virus Challenge. The Journal of Infectious Diseases, 221(Suppl 4), S480-S492.

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