Calcein

Manufactured by Leica

Sourced in Germany

Calcein is a fluorescent dye used in various laboratory applications. It is a chelating agent that binds to calcium ions, producing a green fluorescent signal when exposed to ultraviolet or blue light. Calcein is commonly used to stain and visualize calcium-containing structures, such as cell membranes and bone tissues, in biological samples.

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13 protocols using calcein

Histological and Bone Remodeling Analysis

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Histochemical and immunohistochemical stainings were performed as previously described 30 (link),31 (link). Briefly, dissected bones were fixed in 4% PFA for 48 h, decalcified in 0.5 M EDTA (pH = 7.4) for 4-5 days, and dehydrated in graded ethanol, before being embedded in paraffin. 5-μm-thick bone sections were stained with hematoxylin and eosin (H&E), osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) to quantify number and area of adipocytes, number of osteoblasts, and number of osteoclasts, respectively. Images were photographed using an Olympus CX31 microscope (Olympus, Hamburg, Germany). Primary antibody against OCN and the secondary antibody were purchased from Abcam (Cambridge, Britain). TRAP Staining Kit was obtained from Sigma (St. Louis, USA).
Histomorphometric analysis was conducted to test dynamic bone formation. Briefly, the mice were intraperitoneally injected with 10 mg/kg body weight calcein (Sigma) dissolved in PBS at 10 and 3 days before sacrifice. The collected femurs were fixed in 4% PFA for 48 h, dehydrated in increasing concentrations of ethanol and sectioned without decalcification (60-μm sections). calcein double labeling was observed by a fluorescence microscope (Leica). Mineral apposition rates (MAR) of trabecular bone were determined using Image-Pro Plus 6 software.

Hu Y., Zhang Y., Ni C.Y., Chen C.Y., Rao S.S., Yin H., Huang J., Tan Y.J., Wang Z.X., Cao J., Liu Z.Z., Xie P.L., Wu B., Luo J, & Xie H. (2020). Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism. Theranostics, 10(5), 2293-2308.

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Evaluating Drug Efflux Efficiency via Calcein Assay

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For the estimation of drug efflux efficiency, calcein efflux assay was performed. calceinAM (Invitrogen, Waltham, MA, USA, No. C3099) was administered at the concentration of 1 µg/mL in FluoroBrite^® DMEM (supplemented with 10% FBS and 1% GlutaMAX). Monitoring of free calcein fluorescence intensity was performed with the Leica DMI6000B fluorescence microscope (Alexa488 filter set and time-lapse imaging module (time step = 5 min)). The changes of this parameter over time were used to illustrate the efficiency of drug efflux [17 (link),21 (link)]. For the fluorimetric analyses of calcein-stained specimens, the stacks of fluorescence images of at least 16 randomly chosen confluent culture regions were collected. They were registered with the same excitation/exposure settings (excitation/camera gain/time of exposition). Where indicated, the averaged difference between calcein fluorescence at the beginning and end of experiment (drug efflux intensity) was calculated as the percentage of relevant control.

Warzecha K.W., Pudełek M., Catapano J., Madeja Z, & Czyż J. (2022). Long-Term Fenofibrate Treatment Stimulates the Phenotypic Microevolution of Prostate Cancer Cells In Vitro. Pharmaceuticals, 15(11), 1320.

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Calcein-Based Bone Turnover Analysis

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Mice were injected with Calcein (Sigma) twice: eight days and one day before sacrifice. Femur longitudinal sections were analyzed using an SP8 confocal microscope equipped with a 488 blue laser and HyD detectors (Leica). Pictures of the double Calcein incorporations on the diaphyseal endosteal bone were taken and distances were measured with the LAS X SP8 software (Leica). 2–3 pictures per sample were analyzed and 15 measurements in total per sample were taken.

Scheffler J.M., Grahnemo L., Engdahl C., Drevinge C., Gustafsson K.L., Corciulo C., Lawenius L., Iwakura Y., Sjögren K., Lagerquist M.K., Carlsten H., Ohlsson C, & Islander U. (2020). Interleukin 17A: a Janus-faced regulator of osteoporosis. Scientific Reports, 10, 5692.

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Fluorescent Bone Formation Imaging

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We performed fluorescent double-labeling of the new bone formation with calcein (30 mg/kg, Sigma–Aldrich) and alizarin red (30 mg/kg, Sigma–Aldrich), which were injected 21 and 3 days before the samples were harvested, accordingly. Cranium samples containing CPS or Fe-CPS bioceramic materials were harvested and soaked in 4% paraformaldehyde for 7 days. We performed fluorescent labeling (alizarin red, 543/580–670 nm; calcein, 488/500–550 nm) observation with a confocal laser scanning microscope (Leica, Heidelberg, Germany) after the undecalcified specimens were inserted in polymethyl methacrylate and sliced into 150-mm-thick sections employing a microtome (Leica, Hamburg, Germany). The mineralization rate was simultaneously quantified. Other specimens were decalcified in 10% ethylenediaminetetraacetic acid for 30 days and subsequently inserted in paraffin. After being sliced to a thickness of 5 μm, the sections were mounted on polylysine-coated microscope slides. The sections were then stained with hematoxylin and eosin (H&E) or Masson's trichrome. Finally, we performed morphological analysis using optical microscopy.

Zhang J., Deng F., Liu X., Ge Y., Zeng Y., Zhai Z., Ning C, & Li H. (2022). Favorable osteogenic activity of iron doped in silicocarnotite bioceramic: In vitro and in vivo Studies. Journal of Orthopaedic Translation, 32, 103-111.

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Mitochondrial and Cell Volume Analysis

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4 × 10⁵ RNVCs were plated overnight on 35 mm culture dishes coated with 10 µg/mL laminin, and 24 h later, cells were treated for 6 h with 1 µM or 10 µM of different compounds. Cells were incubated with Mitotracker Red 580 at 200 nM for 20 min at 37 °C, then with 4 μM calcein (Life Technologies, Carlsbad, CA, USA) for 10 min at 37 °C. Z stack images were acquired with a Leica (TCS SP8 gSTED) inverted confocal laser scanning microscope (Mannheim, Germany) equipped with a WLL Laser (495 nm excitation wavelength for calcein and 580 nm for Mitotracker Red 580). Green fluorescence emission was detected with 505–550 nm wide emission slits and 585–700 nm wide emission slits for the red signal under a sequential mode. The pinhole was set at 1.0 Airy unit, and 12-bit numerical images were done with the Leica Application Suite X software (Version 3.5.5; Leica, Wetzlar, Germany).
Mitochondrial network and cell volume 3D model were reconstructed by using the IMARIS software 9.7 version (Bitplane Company, Zurich, Switzerland); consequently, cell volume, mitochondria number, and volume were analyzed using the volume and surface rendering processes.

Liu D., Peyre F., Loissell-Baltazar Y.A., Courilleau D., Lacas-Gervais S., Nicolas V., Jacquet E., Dokudovskaya S., Taran F., Cintrat J.C, & Brenner C. (2022). Identification of Small Molecules Inhibiting Cardiomyocyte Necrosis and Apoptosis by Autophagy Induction and Metabolism Reprogramming. Cells, 11(3), 474.

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Fluorescent Labeling of Bone and Neuromasts in Live Fish

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Live fish were stained with calcein (Sigma Aldrich C0875) to label bone and 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI; Sigma Aldrich D3418) to label canal (CN) and superficial neuromasts (SN) as described in Powers et al. (2018b) (link). For imaging, fish were anesthetized by immersion in ice-cold system water for approximately 15 seconds. Fluorescent images were collected at 7.81x magnification under the GPF (calcein; 488nm) and TXR (DASPEI; 561nm) filters using a Leica stereomicroscope (M205FA) and Leica Application Suite software (LAS v3.8, Wetzlar, Germany). Images were overlaid to visualize bone and neuromasts (Figs. 1, 3 and 5).

Powers A.K., Berning D.J, & Gross J.B. (2020). Parallel evolution of regressive and constructive craniofacial traits across distinct populations of Astyanax mexicanus cavefish. Journal of experimental zoology. Part B, Molecular and developmental evolution, 334(7-8), 450-462.

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Calcein Dye Transfer Assay for GJIC

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Acceptor HUVEC cells grown in Petri dishes were pre-incubated with FF (25 μM) for 4 h and calcein (Life Technologies, C3099)-loaded donor A549 cells/HUVECs were seeded onto the monolayers of HUVEC cells. After 1 h, calcein transfer from donor to acceptor cells was evaluated using a Leica DM IRE2 inverted fluorescence microscope. Gap junctional intercellular coupling (GJIC) was quantified as the percentage of donor cells, which successfully coupled with the acceptor monolayer (coupling index-C_i). Dye transfer from at least 200 donor cells per single coverslip was analyzed in threeindependent experiments [51 (link)].

Piwowarczyk K., Kwiecień E., Sośniak J., Zimoląg E., Guzik E., Sroka J., Madeja Z, & Czyż J. (2018). Fenofibrate Interferes with the Diapedesis of Lung Adenocarcinoma Cells through the Interference with Cx43/EGF-Dependent Intercellular Signaling. Cancers, 10(10), 363.

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Quantifying Cellular Drug Efflux Kinetics

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For the estimation of drug-efflux efficiency, cells were washed twice with warm DPBS before medium replacement for FluoroBrite DMEM (supplemented with 10% FBS and 1% GlutaMAX). calcein-AM (Invitrogen, no. C3099) was administrated at a concentration of 1 µg/ml in the FluoroBrite DMEM. Kinetics of calcein fluorescence intensity that illustrates its efflux were monitored with Leica DMI6000B fluorescence microscope (see above) immediately after calcein-AM administration, using Alexa 488 filter set (excitation—BP 470/40 nm; emission—BP 525/50) and time-lapse imaging module (time step, 5 min; total acquisition time, 60 min). Obtained images were processed with ImageJ software. For the fluorometric analyses of calcein-stained specimens, stacks of fluorescence images of at least 16 randomly chosen confluent culture regions were collected. In each experiment, the stacks were obtained with the same excitation/exposure settings (excitation/camera gain/time of exposition). Fluorescence index was estimated for each stack with LasX software (Leica) and calculated for each specimen [14 ].

Catapano J., Luty M., Wróbel T., Pudełek M., Piwowarczyk K., Kędracka-Krok S., Siedlar M., Madeja Z, & Czyż J. (2022). Acquired drug resistance interferes with the susceptibility of prostate cancer cells to metabolic stress. Cellular & Molecular Biology Letters, 27, 100.

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Quantifying Cell-Cell Coupling

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Donor and acceptor cells were incubated in the presence of DCX/MET for 24 h. Donor cells were then stained with calcein and DiI (Life Technologies; C3099; 5 μM and 10 μM) as described previously [35 (link)] and seeded onto the monolayers of acceptor cells at 1:50 ratio. After 1 h of coincubation of donor and acceptor cells, a transfer of calcein from at least 200 donor cells per coverslip was analyzed using a Leica DMI6000B inverted fluorescence microscope (Leica Alexa 488 filter set) equipped with LasX software. It was further quantified as the percentage of donor cells, which successfully coupled with an acceptor monolayer (coupling index; c_i) and averaged number of coupled acceptor cells/a donor cell (coupling ratio; c_r).

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Gap Junction Intercellular Communication Assay

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GJIC intensity was measured by a fluorescent dye transfer assay as previously described [24] (link) with some modifications. Briefly, donor DU145 cells labelled with calcein and DiI (both from Invitrogen-Life technologies, Carlsbad, USA) were plated (at a ratio of 1:50) onto monolayers of DU145 acceptor cells, 24 h after transfection with PTAI-based lipoplexes. The dynamics of calcein transfer from the donor to acceptor cells was visualized using Leica DMI6000B-AF7000 microscope and the percent of donor cells capable of coupling with at least one acceptor cell within 1 h after seeding was calculated as the coupling index -C i .

Rak M., Ochałek A., Bielecka E., Latasiewicz J., Gawarecka K., Sroka J., Czyż J., Piwowarczyk K., Masnyk M., Chmielewski M., Chojnacki T., Swiezewska E, & Madeja Z. (2016). Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology. The journal of gene medicine, 18(11-12).

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