Calcein am dye

Manufactured by Thermo Fisher Scientific

Sourced in United States

Calcein AM dye is a fluorescent indicator used in cell biology and biochemistry. It is a non-fluorescent, cell-permeable compound that becomes highly fluorescent upon hydrolysis by intracellular esterases, allowing for the detection and quantification of viable cells.

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57 protocols using calcein am dye

Quantifying HUVEC Angiogenesis in TCM

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HUVEC were added to plate pre-coated with Geltrex LDEV-free reduced growth factor basement membrane matrix (Gibco, CA, USA) and incubated with tumor-conditioned medium (TCM) for 15 hours. Tubes formed were stained using calcein-AM dye (Invitrogen, CA, USA) and photographed with Nikon ECLIPSE TS100 inverted fluorescence microscope (Nikon, Tokyo, Japan) using a 5× objective. The images were analyzed using WimTube (Wimasis, Munich, Germany) to quantify angiogenesis.

Ho C.S., Noor S.M, & Nagoor N.H. (2018). MiR-378 and MiR-1827 Regulate Tumor Invasion, Migration and Angiogenesis in Human Lung Adenocarcinoma by Targeting RBX1 and CRKL, Respectively. Journal of Cancer, 9(2), 331-345.

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Fluorescence Recovery After Photobleaching in Cochlear Cultures

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Control and drug-treated organotypic cochlear cultures were incubated for 48 h. Cultures were then washed with phosphate-buffered saline (PBS) and incubated for 5 min at room temperature in 2 μM calcein-AM dye (Invitrogen, catalog# 3100-MP) in DMEM/F12 medium. Cultures were next washed twice with PBS, and fresh DMEM/F12 medium was added. Dye loaded cultures were placed in a live cell incubation chamber (37 °C, 5% CO₂) under a Zeiss LSM800 confocal microscope. To assess the level of gap junction function, both the outer sulcus and inner sulcus regions of organotypic cultures were selected for FRAP analysis. One outer sulcus cell that was adjacent to at least three other cells was photobleached, and the recovery of fluorescence into the photobleached cell was examined every 10 s for eight minutes. Defined regions (288.5 µm² in diameter) of the tightly packed cells of the inner sulcus region were photobleached, and fluorescence recovery to the photobleached region was determined as described above. The intensities of fluorescence were measured in ImageJ, and the percent recovery was calculated as; (F_x − F_P/F_I) × 100, F_x = fluorescence at each time point, F_P = fluorescence after photobleaching, and F_I = initial fluorescence before photobleaching. Area under the curve of a linear regression analysis was calculated using GraphPad Prism 6.

Abitbol J., Beach R., Barr K., Esseltine J., Allman B, & Laird D. (2020). Cisplatin-induced ototoxicity in organotypic cochlear cultures occurs independent of gap junctional intercellular communication. Cell Death & Disease, 11(5), 342.

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Seeding Density and A2-P Effects on ASCs

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ASCs were seeded at different densities (1250, 2500, 5000, 10000 cells/cm²) in basal medium supplemented with various concentrations of A2-P (0, 32.5, 62.5, 125, 250 μM; Sigma, St. Louis, MO). After 24 h for cell attachment, ASCs were stained with the Calcein AM dye (Invitrogen) at room temperature according to the manufacturer’s protocol, and observed under a fluorescent microscope (Leica DMI6000 B). Quantification of the stained cells in the acquired microscopic images was performed by Image J (v1.52k, NIH).

Wu Y.K., Tu Y.K., Yu J, & Cheng N.C. (2020). The Influence of Cell Culture Density on the Cytotoxicity of Adipose-Derived Stem Cells Induced by L-Ascorbic Acid-2-Phosphate. Scientific Reports, 10, 104.

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Cell Migration and Invasion Assay

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Cell migration and invasion assays were done using Boyden chambers as described previously.^{41 (link)} In brief, 2.5 × 10⁴ cells were suspended in base medium devoid of growth factors and placed in each insert coated with or without Matrigel matrix (BD Biosciences). Cells were allowed to move toward full media filled in the bottom well for 24 hours and stained with 4 μg/ml Calcein AM dye (Invitrogen). All Boyden chamber assays were performed at least three times in triplicate.

Whelan K.A., Chandramouleeswaran P.M., Tanaka K., Natsuizaka M., Guha M., Srinivasan S., Darling D.S., Kita Y., Natsugoe S., Winkler J.D., Klein-Szanto A.J., Amaravadi R.K., Avadhani N.G., Rustgi A.K, & Nakagawa H. (2017). Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance. Oncogene, 36(34), 4843-4858.

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Quantifying Cell Migration in Vitro

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In vitro migration assays were performed with modified Boyden chambers using 24-well transwell units with 8 μm pore polycarbonated filters (BD Falcon, Franklin Lakes, NJ). After treatment with either 500 nM of Cediranib or Vandetanib (16 h), cells were dissociated with 0.02% EDTA in PBS, pH 7.4, and 10⁵ cells were resuspended in serum-free medium and transferred to culture inserts (top chamber). The assembly was placed into 24-well plates containing 3 nM CXCL12 (bottom chamber). After 24 hr, migrating cells located at the bottom of the insert were stained with Calcein AM dye (Invitrogen) for 1 h at 37 °C, and analyzed using a fluorescent plate reader for intensity quantification. Cell numbers were determined by comparison to the standard curve for each cell line. Each experiment was performed in triplicate and repeated at least two times using different cell preparations.

Pham K., Luo D., Siemann D.W., Law B.K., Reynolds B.A., Hothi P., Foltz G, & Harrison J.K. (2015). VEGFR inhibitors upregulate CXCR4 in VEGF receptor-expressing glioblastoma in a TGFβR signaling-dependent manner. Cancer letters, 360(1), 60-67.

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Quantitative Wound Healing Assay

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Wound-healing assays were performed as previously described (Platypus Technologies, CMAUFL4)^{49 (link)}. After genome editing, 15,000 cells per well were plated with well inserts in place in culture media. Inserts were then removed the day after plating. Prior to complete wound healing (48–72 h), cells were stained with Calcein AM dye (Invitrogen, C3099) and wound healing was quantified with a fluorescence plate reader (excitation 488 nm/emission 522 nm). Statistical analyses were conducted with one-way ANOVA between groups. Where specific software tools are not named, we used Stata or R for analyses.

Aragam K.G., Jiang T., Goel A., Kanoni S., Wolford B.N., Atri D.S., Weeks E.M., Wang M., Hindy G., Zhou W., Grace C., Roselli C., Marston N.A., Kamanu F.K., Surakka I., Venegas L.M., Sherliker P., Koyama S., Ishigaki K., Åsvold B.O., Brown M.R., Brumpton B., de Vries P.S., Giannakopoulou O., Giardoglou P., Gudbjartsson D.F., Güldener U., Haider S.M., Helgadottir A., Ibrahim M., Kastrati A., Kessler T., Kyriakou T., Konopka T., Li L., Ma L., Meitinger T., Mucha S., Munz M., Murgia F., Nielsen J.B., Nöthen M.M., Pang S., Reinberger T., Schnitzler G., Smedley D., Thorleifsson G., von Scheidt M., Ulirsch J.C., Arnar D.O., Burtt N.P., Costanzo M.C., Flannick J., Ito K., Jang D.K., Kamatani Y., Khera A.V., Komuro I., Kullo I.J., Lotta L.A., Nelson C.P., Roberts R., Thorgeirsson G., Thorsteinsdottir U., Webb T.R., Baras A., Björkegren J.L., Boerwinkle E., Dedoussis G., Holm H., Hveem K., Melander O., Morrison A.C., Orho-Melander M., Rallidis L.S., Ruusalepp A., Sabatine M.S., Stefansson K., Zalloua P., Ellinor P.T., Farrall M., Danesh J., Ruff C.T., Finucane H.K., Hopewell J.C., Clarke R., Gupta R.M., Erdmann J., Samani N.J., Schunkert H., Watkins H., Willer C.J., Deloukas P., Kathiresan S, & Butterworth A.S. (2022). Discovery and systematic characterization of risk variants and genes for coronary artery disease in over a million participants. Nature Genetics, 54(12), 1803-1815.

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Celigo-based Cytolytic Activity Assay

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The Celigo Imaging Cytometer based cytolytic assay was performed to determine the cytolytic activity of transduced NK cells at 4 h, as previously described.⁶⁴ ^,⁶⁵ (link) Briefly, B7H3⁺ targets were labeled with Calcein AM dye (Invitrogen). After staining, targets were cocultured with transduced and UT control NK cells in a flat-bottom microplate at E:T ratios of 10:1, 5:1, and 1:1. Live cells were detected by imaging plates after 4 h of incubation using the Celigo Imaging Cytometer system. “Target lysis” was calculated according to the manufacturer’s recommendations as follows:

% T a r g e t l y s i s = 1 - \frac{# Calcein AM Target cells with Effector cell}{# Calcein AM Target cells without Effector cell} \times 100

Chaudhry K., Geiger A., Dowlati E., Lang H., Sohai D.K., Hwang E.I., Lazarski C.A., Yvon E., Holdhoff M., Jones R., Savoldo B., Cruz C.R, & Bollard C.M. (2022). Co-transducing B7H3 CAR-NK cells with the DNR preserves their cytolytic function against GBM in the presence of exogenous TGF-β. Molecular Therapy. Methods & Clinical Development, 27, 415-430.

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T98G Cell Viability Assessment

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T98G cell viability was measured using the Calcein AM assay, where 5 × 10³ cells were plated per well in a 96-well dish. Cells were treated with 680C91 (10 and 20 μM) for 24 h. Cells were treated for with varying concentrations of BCNU (0–2 mM) for an additional 48 h before incubation with Calcein AM dye (2 μM) (Invitrogen; Cat# C1430, Grand Island, NY) at 25°C for 30 m. To determine percent viability, fluorescence values were obtained using Synergy4 plate reader at an excitation wavelength 485 nm and emission wavelength 528 nm. The EC₅₀ values were calculated by fitting the resulting data to a four-parameter logistic model using Prism software.

Reed M.R., Maddukuri L., Ketkar A., Byrum S.D., Zafar M.K., Bostian A.C., Tackett A.J, & Eoff R.L. (2021). Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells. NAR Cancer, 3(2), zcab014.

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Calcein-AM Staining of Viable Cells

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Adherent cells were stained using a calcein-AM dye (Invitrogen). Cells were aspirated and washed with PBS, followed by incubation with 2 μM calcein-AM in PBS for 15 min. Viable adherent cells were stained green. Immunofluorescence images were acquired using a fluorescence microscope (IX-70, Olympus) equipped with a color CCD camera (Optronics MagnaFire).

Lee J.Y, & Schmidt C.E. (2014). Amine-functionalized polypyrrole: inherently cell adhesive conducting polymer. Journal of biomedical materials research. Part A, 103(6), 2126-2132.

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Lassa Virus Fusion Assay

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The effector Cos7 cells were transfected with the Lassa virus GPc expression vector. Briefly, cells were grown on 35 mm culture dishes to ~60% confluency and transfected with 4 μg GPc expression vector using a calcium-phosphate protocol [22 (link)]. After 48 hours following transfection, cells were loaded with 1.3 μM of the green cytoplasmic Calcein-AM dye (Invitrogen). In parallel, 293T cells or their derivatives stably expressing human IFITM1, IFITM2 or IFITM3 [22 (link)] were labeled with 30 μM of the blue cytoplasmic dye CMAC (Invitrogen). Effector and target cells were washed, detached from the culture dishes using a non-enzymatic solution, resuspended in PBS++, mixed at a 1:1 ratio and co-plated onto 8-well chamber slides. After incubating for 30 min at room temperature, cells were exposed to a pH 5.0 buffer at 37°C for 20 min, and the resulting cell-cell fusion was measured by visual inspection under a fluorescent microscope, as described in [22 (link)]. Ten fields of view each containing 10–12 heterologous cell pairs were examined in each well.

Suddala K.C., Lee C.C., Meraner P., Marin M., Markosyan R.M., Desai T.M., Cohen F.S., Brass A.L, & Melikyan G.B. (2019). Interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes. PLoS Pathogens, 15(1), e1007532.

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