Calcein green am

Manufactured by Thermo Fisher Scientific

Sourced in United States, Italy

Calcein green AM is a fluorescent dye used as a cell viability indicator in biological research. It is a cell-permeant dye that is hydrolyzed by intracellular esterases to produce a green fluorescent product, which can be detected using appropriate excitation and emission wavelengths.

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46 protocols using calcein green am

Quantifying Cellular Morphology of BMMs

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BMMs were cultured and synchronized using cell culture media containing 0.1% fetal bovine serum (FBS). CellTrace Calcein Green AM (Molecular Probes, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA), a cell-permeant dye was added for labeling viable cells during the experiment in an order to acquire size and shape of both WT and AR-null BMMs. Calcein Green AM (Molecular Probes, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) is a fluorogenic cell-permeant dye that can be used to determine cell viability and morphology. In living cells, the nonfluorescent Calcein Green AM (Molecular Probes, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) is converted to an intensely green-fluorescent calcein after intracellular esterases remove the acetoxymethyl (AM) esters. The cells were visualized using Evos, AMG microscope (ThermoFisher Scientific, Grand Island, NY). Briefly, 100 × 100 μm grids having cells were selected to measure the area accurately and the differences in size (cell-area) were measured using the ‘Magic Wand” tool (Image J software).

Singh M., Kapoor A., McCracken J., Hill B, & Bhatnagar A. (2017). Aldose reductase (AKR1B) deficiency promotes phagocytosis in bone marrow derived mouse macrophages. Chemico-biological interactions, 265, 16-23.

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Quantifying Parasite Invasion Dynamics

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Red-green invasion assays were performed as described previously, with the following modifications (37 (link)). Parasites were filter purified and resuspended to 5 × 10⁷/ml in DMEM–3% FBS–10 mM HEPES (D3). Calcein green AM (Invitrogen) was added to the parasites to a final concentration of 1 µM and incubated for 15 min with agitation in the dark. Parasites were then centrifuged at 1,000 × g and resuspended in fresh D3, and 1 × 10⁷ parasites were allowed to invade subconfluent host cell monolayers in eight-well chamber slides for 20 min before fixation. Slides were stained with anti-SAG1 antibodies and Alexa594 secondary antibodies to differentiate extracellular and intracellular parasites. For timed invasion assays, 2.5 × 10⁵ parasites were inoculated into each well of a chamber slide and the slide was fixed at a defined time point. For the cytochalasin D attachment assay, parasites were incubated with 1 µM Calcein green AM (Life Technologies) for 20 min before the addition of 2 µM cytochalasin D for 10 min at room temperature. Parasites were centrifuged at 1,000 × g for 10 min, resuspended in D3 and 1 µM cytochalasin D, inoculated onto host cell monolayers, and allowed to attach for 20 min. Slides were fixed and enumerated on the basis of calcein green fluorescent parasites.

Huynh M.H, & Carruthers V.B. (2016). A Toxoplasma gondii Ortholog of Plasmodium GAMA Contributes to Parasite Attachment and Cell Invasion. mSphere, 1(1), e00012-16.

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Quantifying Intracellular Esterase Activity

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Calcein-AM indicates intracellular esterase activity. Cells were washed twice with Phosphate buffer saline (PBS). Cells were next stained with the 2 μM of calcein-green-AM (Molecular Probes, Invitrogen, Leiden NL) for 45 min at 37° 5% CO₂ according to manufacturers instructions. They were then immediately analyzed by FACS on a SORP FACSAria2 (BD Bioscience, San Jose, CA) as described.

Pasquier J., Vidal F., Hoarau-Véchot J., Bonneau C., Daraï E., Touboul C, & Rafii A. (2018). Surgical peritoneal stress creates a pro-metastatic niche promoting resistance to apoptosis via IL-8. Journal of Translational Medicine, 16, 271.

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Phagocytosis Kinetics of Macrophages

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WT-BMMs and AR-null BMMs were seeded in 6-well plates, and growth synchronized in 0.1% fetal bovine serum (FBS) medium a day before. After 24 h, the cells were treated with or without LPS (100 μM) for 12 h and then incubated the latex beads coated with phycoerythrin (PE) labeled Rabbit-IgG. After 6 h of incubation, the cells were harvested for measuring the kinetics of phagocytosis using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Fluorescence microscopy of the macrophages was also carried out after platting them in complete growth medium. Intense red colored particles ingested by phagosomes were photographed. For assessing the phagocytosis under microscope, Calcein Green, AM (Molecular Probes, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) was used to capture live cell images to detect engulfed fluorescent beads and DAPI (Sigma, St. Louis, MO, USA) was used to stain the nuclei. Pictures were taken using Evos, AMG microscope (ThermoFisher Scientific, Grand Island, NY).

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Comprehensive Cellular and Biochemical Assays

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All reagents used were of analytical grade and were purchased from the respective vendors as indicated. Alpha tubulin antibody, LPS, TPP tissue culture flasks, 10 cm petridishes, and 6-well plates (Sigma, St. Louis, MO, USA), AR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ATP assay kit, NADP/NADPH assay kit (Abcam, Cambridge, MA, USA), Calcein Green, AM (Molecular Probes, Life Technologies, ThermoFisher Scientific, Waltham, MA, USA), Enhanced chemiluminescence (ECL plus) reagents were from Pierce (Rockford, IL, USA), Fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, USA), Gentamicin solution, Hepes, Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG F(ab′)₂ (Cell Signaling, Beverly, MA, USA), Phagocytosis assay kit (Cayman Chemical, Ann Arbor, MI, USA), Protein assay kit (Biorad, Hercules, CA, USA), RPMI-1640 medium (1×) with 2.05 mM L-glutamine and 2.0 g/L glucose (Hyclone Laboratories, GE Healthcare Life Sciences, South Logan, UT, USA), Phosphate-buffered saline were from (Gibco, Life Technologies, Waltham, MA, USA).

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Cytotoxicity Assay with Stimulated PBMCs

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Target cells were labelled with 15 μM Calcein Green AM (Molecular Probes, Darmstadt), washed, and mixed with ex vivo expanded and stimulated PBMCs from healthy unrelated donors in RPMI 1640 GlutaMAX™ supplemented with 10 % FBS and Penicillin (100 U/mL)/Streptomycin (100 μg/mL) (Gibco, Thermo Fisher Scientific, Darmstadt) at an E : T ratio of 10 : 1. Antibody derivatives were diluted to the desired concentration with medium and added to 200 μL reaction volumes in a 96-well round bottom tissue culture plate (CellStar, Greiner bio-one). After a 3 hour incubation period at 37°C, 5 % CO₂, 100 μL supernatant was transferred to a black 96-well flat bottom plate (Nunc) and fluorescence was determined on a Berthold Mithras plate reader (Berthold Technologies, Bad Wildbad). Maximum lysis was achieved by addition of 2.5 % Triton X-100. Specific lysis was calculated as follows:
% Specific Lysis = 100 * [( RLU (sample) – RLU (background)) / (RLU (max. lysis) – RLU (background))], where RLU = relative light units and the background is the degree of lysis obtained with effector cells alone in the absence of added triplebody.

Roskopf C.C., Braciak T.A., Fenn N.C., Kobold S., Fey G.H., Hopfner K.P, & Oduncu F.S. (2016). Dual-targeting triplebody 33-3-19 mediates selective lysis of biphenotypic CD19+ CD33+ leukemia cells. Oncotarget, 7(16), 22579-22589.

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Calcein Imaging of Olfactory Bulb

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Animals were killed (see above) 1, 2, 3 and 7 days after unilateral nerve transection and a whole mount preparation of the OB was prepared from excised tissue blocks (as described above). The preparation was incubated in bath solution (see “Solutions” section) containing 100 μM Calcein green/AM (Molecular Probes, Thermo Fisher Scientific), 30 μM Alexa 594 dextran (10,000 MW, Molecular Probes, Thermo Fisher Scientific), and 50 μM MK571 (Sigma-Aldrich), an inhibitor of multidrug resistance transporters. Calcein green/AM was initially dissolved in DMSO (Sigma-Aldrich) and Pluronic F-127 (Molecular Probes, Thermo Fisher Scientific). After an incubation time of 2 h, a series of image stacks of the whole ventral OB was obtained using multiphoton microscopy. Relative changes of the OB volume of the transected side to the non-transected side were compared.

Hawkins S.J., Weiss L., Offner T., Dittrich K., Hassenklöver T, & Manzini I. (2017). Functional Reintegration of Sensory Neurons and Transitional Dendritic Reduction of Mitral/Tufted Cells during Injury-Induced Recovery of the Larval Xenopus Olfactory Circuit. Frontiers in Cellular Neuroscience, 11, 380.

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Quantifying Cell Death via Fluorescence

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Cultures were incubated under the designated experimental conditions for 30 minutes at 37 °C, then transferred to fresh BSS and incubated for an additional 24 hours. All incubations were in 0% CO₂ (room air). At 24 hours calcein green-AM (5 µM, Molecular probes) was added to identify living cells, and Hoechst 33258 (1 µM, Molecular probes) to identify all cell nuclei. Analysis at the 24 hour time point was done to preclude the possibility of calcein leakage from viable cells through transiently opened pores. Photographs were taken of 3 randomly determined fields of each culture well, and cell death was quantified by counting the number of cells labeled by Hoechst 33258 but not calcein green^{27 (link)}. Cells were counted in 3 fields from a minimum of 4 wells per plate in each independent experiment.

Minnella A.M., Zhao J.X., Jiang X., Jakobsen E., Lu F., Wu L., El-Benna J., Gray J.A, & Swanson R.A. (2018). Excitotoxic superoxide production and neuronal death require both ionotropic and non-ionotropic NMDA receptor signaling. Scientific Reports, 8, 17522.

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Visualizing Fibrin and Platelets in Clots

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To visualize distinctly fibrin and platelets in a fluorescent confocal microscope, Alexa-Fluor 594-labeled human fibrinogen (Molecular Probes, Grand Island, NY, 40 µg/ml final concentration) and calcein green, AM (Molecular Probes, 0.2 µg/ml final) were added to PRP samples and incubated for 10 min at 37 °C before initiation of clotting. Clotting was induced by adding CaCl₂ (40 mM final concentration) and thrombin (0.75 or 1 U/ml final concentration) to PRP containing the fluorescent probes. Thirty microliters of samples was quickly transferred onto 35 × 10 mm PELCO clear wall glass bottom cell culture dishes in the environmental chamber of the confocal microscope for the time course z-stack imaging. In some experiments, the glass was pre-coated with 4% (v/v) Triton X-100 in PBS to prevent attachment of fibrin and allow for unconstrained clot contraction. In inhibitory experiments abciximab (ReoPro; Eli Lilly, Indianapolis, IN, USA) was added to PRP at 100 µg/ml or blebbistatin (Sigma-Aldrich, St. Louis, MO, USA) was used at 300 µM (both final concentrations) prior to the fluorescent probes and incubated for 15 min at 37 °C.

Kim O.V., Litvinov R.I., Alber M.S, & Weisel J.W. (2017). Quantitative structural mechanobiology of platelet-driven blood clot contraction. Nature Communications, 8, 1274.

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Mitochondrial Permeability Transition Pore Imaging

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The opening of the mPTP was determinate in cells previously treated with 50 mM cobalt chloride for 15 min, before incubation with 1 μM Calcein Green AM (Molecular Probes, OR, USA) and 0.5 μM Mitotracker Red (Molecular Probes, OR, USA) in KRH-glucose buffer at 37 °C for 30 min [32] (link). Quenching of free Calcein by cobalt chloride allowed the preservation of mitochondrial integrity as an mPTP indicator [18] (link), [31] . Quantification of fluorescence intensity for three separate experiments was carried out by analyzing the Calcein intensity in 25 images for every indicated condition using Image J software.

Pérez M.J., Ponce D.P., Aranguiz A., Behrens M.I, & Quintanilla R.A. (2018). Mitochondrial permeability transition pore contributes to mitochondrial dysfunction in fibroblasts of patients with sporadic Alzheimer's disease. Redox Biology, 19, 290-300.

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