Celltracker red

Manufactured by Thermo Fisher Scientific

Sourced in United States

CellTracker Red is a fluorescent dye used for labeling living cells. It is cell-permeant and gets metabolically converted into a cell-impermeant fluorescent form, enabling long-term tracking of labeled cells. The dye exhibits red fluorescence and can be detected using standard fluorescence microscopy or flow cytometry techniques.

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Close spelling variants of this product

CellTracker Red CMTPX (137 citations) CellTracker Red CMTPX Dye (105 citations) CellTracker Deep Red (95 citations) CellTracker Deep Red Dye (74 citations) CellTracker™ Red CMPTX (22 citations) CellTracker™ Red dye (11 citations) CellTracker™ Red CMPTX Dye (8 citations) Celltracker red and green (6 citations) CellTracker™ Deep Red (CTDR) (3 citations) CellTrackers (2 citations)

109 protocols using celltracker red

Mitochondrial Transfer in Co-Culture

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MMC-hWJSCs and CD34+ cells were stained with two different color-coded dyes for ease of identification under confocal microscopy. MMC-hWJSCs were incubated with 10 μM of CellTracker™-Red (Invitrogen) for 30 min at 37 °C in a 5% CO₂ in air atmosphere while the CD34+ cells were stained with 10 μM of MitoTracker™-Green (Invitrogen) at 37 °C for 30 min. The cells for both groups were centrifuged and the media discarded. The cell pellets were washed with PBS before incubation for another 30 min with their own growth media. The CellTracker™-Red-MMC-hWJSCs and MitoTracker™-Green-CD34+ cells were then co-cultured in a Lab-Tek^(R) Chambered #1.0 Borosilicate cover glass system for 24 h (Thermo Scientific). The co-culture platform was performed in Stemspan SFEM serum-free medium that contains Iscove’s modified Dulbecco medium with 4500 mg/L of d-glucose and a CC110 cytokines cocktail (Stem Cell Technologies, Singapore Pte Ltd). The pH of the co-culture environment was neither extremely acidic nor alkaline as it readily supported the growth of the CD34+ cells according to the manufacturer’s recommendations. An Olympus FluoView FV1000 laser scanning confocal microscope (Olympus, Japan) was then used to analyze the transfer of mitochondria from CD34+ cells to MMC-hWJSCs.

Lin H.D., Fong C.Y., Biswas A, & Bongso A. (2020). Allogeneic human umbilical cord Wharton’s jelly stem cells increase several-fold the expansion of human cord blood CD34+ cells both in vitro and in vivo. Stem Cell Research & Therapy, 11, 527.

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3D Angiogenic Sprouting Imaging

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To monitor 3D angiogenic sprouting, Z-stack images were acquired with a Zeiss LSM 710 or 880 Laser Scanning Confocal Microscopes. All stacked images were reconstructed by using ZEN lite software (Zeiss) and ImageJ (NIH) for 3D visualization. Prior to imaging EC-macrophage-tumor cell interactions, RAW264.7 cells were labeled with Hoechst 33342 (5 μg/mL) and GBM cells were labeled with Cell Tracker Red (5 μM; C34552, Thermo Fisher-Scientific). For other experiments, RAW264.7 cells were labeled with Cell Tracker Red (5 μM; C34552, Thermo Fisher-Scientific).

Cui X., Tan Morales R.T., Qian W., Wang H., Gagner J.P., Dolgalev I., Placantonakis D., Zagzag D., Cimmino L., Snuderl M., Lam R.H, & Chen W. (2018). Hacking Macrophage-associated Immunosuppression for Regulating Glioblastoma Angiogenesis. Biomaterials, 161, 164-178.

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ROS Measurement in Responder Cells

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ROS was measured using the ROS-ID kit (Enzo Life Sciences) according to the manufacturer’s protocol. Live cells were also stained with Cell Tracker Red (CTR; Molecular Probes) as a control for cell volume. Responder cells were seeded into chamber slides, incubated for 24 h with EV in media +1% BSA, stained with CTR (1:500, 30 min in media) followed by DCFDA (1: 5000, 45 min in PBS), washed with PBS, and then imaged. Live cell images were captured using an Olympus FV1000 spectral confocal microscope for CTR (Channel 3) and DCFDA (Channel 1). FIJI (ImageJ) software was used to create masks from channel 3 (CTR), and then the masks were transferred onto channel 1 (DCFDA). The mean gray values (MGVs) in channels 1 and 3 were recorded within the masks, and the DCFDA/CTR MGV ratio was calculated for each mask. See Figure 3B for plots of ranked DCFDA/CTR ratios of individual cells from representative experiments. From each experiment, we collected the ratios from at least 200 cells per sample. We then determined the median and the interquartile range of ratios collected from one to three experiments (200–600 cells) as indicated in the figure legends.

Rastogi S., Hwang A., Chan J, & Wang J.Y. (2018). Extracellular vesicles transfer nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander effects. Molecular Biology of the Cell, 29(18), 2228-2242.

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Tracking Adoptive NK Cell Trafficking

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Bulk idNK cell preparations were labeled with 1 μM of fluorescence dye “cell-tracker red” CMTPX (Molecular Probes^® –Life Technologies, Grand Island, NY, USA) for 15 min at 37°C followed by 30 minutes incubation in pre-warmed complete RPMI media (without IL-15) following manufacturer’s instructions. The cells were injected at doses of 2 x10⁶, 5 x10⁶ and 15 x10⁶ cells by tail vein injection in pregnant Rag2^-/- γc ^-/- females at gd9 and mice euthanized at gd12. Non-pregnant mice received 15 x10⁶ cells and were euthanized three days post injection. Liver, lung, spleen and implantation sites were collected, embedded in O.C.T Compound Medium (Tissue-Tek® 4583) and immediately placed at -80°C. Frozen blocks were stored at -20C. 7 mm sections were cut using cryostat (Shandon Cryotome, Thermo Scientific®) at -20°C and placed on adhesion slides (Superfrost Plus, Thermo Scientific®). Images were acquired with a fluorescence microscope (Olympus BX41—Cellsens® Standard—Life Science Imaging Software). Images from the DAPI, FITC, and Texas Red channels were obtained and overlaid.

Cavalli R.C., Cerdeira A.S., Pernicone E., Korkes H.A., Burke S.D., Rajakumar A., Thadhani R.I., Roberts D.J., Bhasin M., Karumanchi S.A, & Kopcow H.D. (2016). Induced Human Decidual NK-Like Cells Improve Utero-Placental Perfusion in Mice. PLoS ONE, 11(10), e0164353.

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Sorting and Analyzing Hematopoietic Cell Populations

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Jurkat (CRL-1990) and K562 (CCL-243) cells were purchased from ATCC. The cells were cultured and maintained in RPMI-1640 medium (Sigma) with the addition of 10% FBS (Atlanta Biologicals). All cells were incubated at 37 °C with 5% CO₂. Cells were expanded to 80% confluency in cell culture flasks over two days. White blood cells (WBCs) were collected from fresh whole blood collected from deidentified donor. Centrifugation followed by red blood cell lysis buffer (Alfa Aesar) was employed to eliminate red blood cells. The isolated white blood cells were resuspended in PBS. To differentiate cell types in flow cytometry (Accuri C6, BD), WBCs were labeled with 2 μM with CellTracker™ red and Jurkat cells in green (Molecular Probes, Inc.) for approximately 1 h at 37 °C. After labeling the cells with the dye, the accuracy of sorting could be quantified using sensitivity analysis. For studies of the effect of chemotherapy treatment on sorting, all cells were treated with daunorubicin at concentrations of 0.05 μM for 15 h, which was found to induce apoptosis in a vast majority of K562 and Jurkat cells and cause stiffening.³⁵ (link) From flow cytometry analysis of the sorted subpopulations, the enrichment factor was calculated using the following equation:

\frac{{(Number o f X cells / Number o f Y cells)}_{Outlet}}{{(Number o f X cells / Number o f Y cells)}_{Inlet}} .

Islam M., Raj A., McFarland B., Brink H.M., Ciciliano J., Fay M., Myers D.R., Flowers C., Waller E.K., Lam W., Alexeev A, & Sulchek T. (2020). Stiffness based enrichment of leukemia cells using microfluidics. APL Bioengineering, 4(3), 036101.

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Bioinks for 3D Printed Tissue Constructs

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P4-P6 human dermal fibroblasts (Cell Applications, Inc.) and human umbilical vein endothelial cells (Lonza Inc.) were incorporated into 5 w/v% gelatin or 3 w/v% fibrinogen at PEGX ratios of 0.1 and 0.2 respectively. Cell homogeneity throughout the bioink was assessed by sequentially collecting extruded bioink into tared microtubes. Wet weight was collected, gels were degraded with proteinase K (Sigma), and the solutions were analyzed for DNA content by the Picogreen assay (Life Technologies) using a microplate reader. Cell viability was assessed with the Live/Dead® assay (Life Technologies) according to manufacturer’s instructions. P4-P6 bone marrow-derived human mesenchymal stem cells (Lonza, Inc.) were seeded onto HUVEC encapsulated PEG-gelatin printed constructs. HUVECs and hMSCs were labeled with CellTracker^™ Red (Molecular Probes®) and CellTracker^™ Green, respectively according to manufacturer’s instructions. Cells were imaged with a Nikon C2+ confocal and Nikon AZ 100 fluorescent stereoscope.

Rutz A.L., Hyland K.E., Jakus A.E., Burghardt W.R, & Shah R.N. (2015). A Multi-Material Bioink Method for 3D Printing Tunable, Cell-Compatible Hydrogels. Advanced materials (Deerfield Beach, Fla.), 27(9), 1607-1614.

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In Vitro Angiogenesis Assay with PMSC and HUVEC Co-culture

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Aliquots of 50 μl Matrigel (Corning) were plated into each well of 96-well plates (Corning) and polymerized for 30 min at 37 °C. The PMSCs labeled with CMFDA (cell tracker green Molecular Probes), EC-differentiated PMSCs labeled with CMFDA, and HUVECs labeled with CMTPX (cell tracker red, Molecular Probes) were resuspended at 10⁶ cells/ml. The cell suspension (100 μl) was added, respectively, to each well and incubated at 37 °C for 4 h.
For tube formation assay in the HUVECs and PMSCs coculture system, 50 μl CMFDA-labeled PMSCs or CMFDA-labeled EC-differentiated PMSCs suspension, together with 50 μl CMTPX-labeled HUVECs suspension, was added to each well and incubated at 37 °C for 4 h. The tube formation was visualized under phase-contrast microscopy (Olympus, Tokyo, Japan) and the ratio of green/red fluorescence intensity was calculated by ImageJ software.

Zhang Y., Zhong Y., Liu W., Zheng F., Zhao Y., Zou L, & Liu X. (2022). PFKFB3-mediated glycometabolism reprogramming modulates endothelial differentiation and angiogenic capacity of placenta-derived mesenchymal stem cells. Stem Cell Research & Therapy, 13, 391.

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Multicellular Spheroid Formation Methodology

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Human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells (HASMC) and human normal dermal fibroblasts (HNDFB) were purchased from Lonza, Inc. (Walkersville, MD, USA). HUVEC, HASMC and HNDFB were cultured in an appropriate medium, i.e., endothelial cell medium (ECM), smooth muscle cell medium (SMCM), fibroblast medium (FM), respectively, with growth supplement (Lonza). The cells were passaged every 3 days and were used within the third to fifth passage in this study. The cells were cultured on 0.5% gelatin-coated dishes (Techno Plastic Products, Sigma-Aldrich) and were maintained at 37°C in a humidified atmosphere containing 5% CO₂. HUVEC were incubated with CellTracker Red (10μM) (Molecular Probes, Invitrogen Corp, Carlsbad, CA) for 30 minutes to allow visualization of the HUVEC in MCSs. Mixed cell suspensions (a total cell count of 2.5 x 10⁴) composed of HUVEC (40%), HASMC (10%), and HNDFB (50%) was plated into each well of ultra-low attachment round-shaped 96-U-well plates (Sumitomo Bakelite, Tokyo, Japan) filled with triple mixed media of ECM, SMCM, and FM with a ratio of 1:1:1. After 24 hours, the cells aggregated to form a round shaped MCS. According to the automated measurement function equipped on our Bio-3D printer, the size of the MCS was 615.0±51.3 μm (mean ±SD) for 500 MCSs.

Itoh M., Nakayama K., Noguchi R., Kamohara K., Furukawa K., Uchihashi K., Toda S., Oyama J.I., Node K, & Morita S. (2015). Scaffold-Free Tubular Tissues Created by a Bio-3D Printer Undergo Remodeling and Endothelialization when Implanted in Rat Aortae. PLoS ONE, 10(9), e0136681.

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Macrophage-Mediated Phagocytosis Assay

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Idelalisib was added to plated macrophages in AIM-V media and incubated at 37°C for 1 h, followed by the addition of rituximab or obinutuzumab. WIL2-S target cells were labeled with CellTracker Red (CTR; Molecular Probes; Thermo Fisher Scientific), as per the manufacturer’s protocol, and combined with the macrophages at an E:T ratio of 3:1. The cocultures were incubated for 2 h at 37°C. Cells were then stained with FITC anti-CD14 (BD Biosciences) and FITC anti-CD11b (eBioscience) and analyzed on an LSR II cytometer (BD Biosciences).
Cells within the live cell gate containing CD14⁺ M1 or M2c macrophages and CTR⁺ target cells were further separated within a dot plot quadrant. FITC⁺ effector macrophages were placed in the upper left quadrant of the y-axis, and CTR⁺ target cells were placed in the lower right quadrant of the x-axis. Double-positive cells (FITC⁺CTR⁺) in the upper right quadrant represent phagocytized target cells, and the percentage of phagocytosis was calculated as (% double-positive cells + [% double-positive cells + % target cells alone]) × 100.

Palazzo A., Herter S., Grosmaire L., Jones R., Frey C.R., Limani F., Bacac M., Umana P., Oldham R.J., Marshall M.J., Cox K.L., Turaj A.H., Cragg M.S., Klein C., Carter M.J, & Tannheimer S. (2018). The PI3Kδ-Selective Inhibitor Idelalisib Minimally Interferes with Immune Effector Function Mediated by Rituximab or Obinutuzumab and Significantly Augments B Cell Depletion In Vivo. The Journal of Immunology Author Choice, 200(7), 2304-2312.

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Melanoma Cell Uptake and Viability Assay

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100 μl of concentrated A375 melanoma cell solutions (≥ 10⁵ cells/ml) was transferred to a glass bottom well plate (384 PerkinElmer® cell carrier). After 4 hours, unattached cells and media were aspirated and the wells were refilled with 100 μl of fresh RPMI media supplemented with 10% FBS. In each well, the microgel sensors (synthesized with 0.1 mM, 0.4 mM, or 4 mM sensor peptides) or soluble sensor peptides (0.1 mM, 1 mM) were added. To test cellular uptake of sensor molecules, bright-field and z-stack fluorescent images were collected immediately after the addition of sensor molecules and again at one-day after.
To test cell viability, dead cells were identified by ethidium homodimer uptake (Molecular Probes). After 24 hours of incubation with the sensor molecules, cell culture media was aspirated, and the A375 cells were exposed to the ethidium homodimer solution (2 μl/ml) for 30 min, and then rinsed in RPMI media. The total number of cells were identified by the bright-field image, and the number of dead cells were identified by the red fluorescence of DNA-bound ethidium homodimer. After 3 days of incubation, cells were labelled with CellTracker^™ Red (Molecular Probes), and cellular morphology and number were quantified from the fluorescence images by Harmony® software.

Shin D.S., Tokuda E.Y., Leight J.L., Miksch C.E., Brown T.E, & Anseth K.S. (2017). Synthesis of microgel sensors for spatial and temporal monitoring of protease activity. ACS biomaterials science & engineering, 4(2), 378-387.

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