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Cell Explorer™ Fixable Live Cell Tracking Kit *Green Fluorescence*

OverviewpdfSDSpdfProtocol


Our Cell Explorer™ fluorescence imaging kits are a set of tools for labeling cells for fluorescence microscopic investigations of cellular functions. The effective labeling of cells provides a powerful method for studying cellular events in a spatial and temporal context. This particular kit is designed to uniformly label live cells in green fluorescence for the studies that require the fluorescent tag molecules retained inside cells for relatively longer time. The cells can be fixed to retain the imaging pattern. The kit uses a non-fluorescent dye that carries a cell-retaining moiety. The dye becomes strongly fluorescent upon entering into live cells, and trapped inside live cells to give a stable fluorescence signal for relatively long time. The dye is a hydrophobic compound that easily permeates intact live cells. The labeling process is robust, requiring minimal hands-on time. It can be readily adapted for a wide variety of fluorescence platforms such as microplate assays, immunocytochemistry and flow cytometry. It is useful for a variety of studies, including cell adhesion, chemotaxis, multidrug resistance, cell viability, apoptosis and cytotoxicity. The kit provides all the essential components with an optimized cell-labeling protocol.

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC filter
EmissionFITC filter
Recommended plateBlack wall/clear bottom

Components


Component A: Track It™ Green1 vial
Component B: Assay Buffer1 bottle (20 mL)
Component C: DMSO1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare samples
  2. Add Track It™ Green working solution
  3. Stain the cells at 37°C for 15 to 30 minutes
  4. Wash the cells
  5. Examine the specimen under fluorescence microscope with FITC filter (Ex/Em = 490/520 nm) or flow cytometer with 530/30 nm filter (FITC channel) 
Important      Thaw all the components at room temperature before opening.

CELL PREPARATION

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Track It™ Green stock solution (1000X)
Add 25 µL of DMSO (Component C) into the vial of Track It™ Green (Component A) and mix well to make 1000X Track It™ Green stock solution.
Note     The unused portion of 1000X Track It™ stock solution should be stored at -20 oC. Avoid repeated freeze/thaw cycles.

PREPARATION OF WORKING SOLUTION

Track It™ Green working solution
Dilute 1000X Track It™ Green stock solution into Assay Buffer (Component B) at 1:1000 ratio to make Track It™ Green working solution.
Note     The final concentration of the Track It™ Green should be empirically determined for different cell types and/or experimental conditions. In general, long-term staining (more than about 3 days) or the use of rapidly dividing cells will require 1:500 dilution to double the dye concentration. Dye at a lower concentration up to 1:2000 dilution may be needed for shorter experiments, such as viability assays. To maintain normal cellular physiology and reduce potential artifacts, the concentration of the dye should be kept as low as possible.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Remove Growth medium, wash cells with PBS once.
  2. Add 100 µL Track It™ Green working solution to each well.
  3. Incubate the cells in a 37 °C, 5% CO2 incubator for 15 to 60 minutes.
  4. Wash cells with Hanks and 20 mM Hepes buffer (HHBS) or an appropriate buffer.
  5. Fill the cell wells with Assay Buffer or  an appropriate buffer.
  6. Image the cells using a fluorescence microscope with FITC filters (Ex/Em = 490/520 nm) or monitor the fluorescence intensity with a flow cytometer using 530/30 nm emission filter (FITC channel). Gate on the cells of interest, excluding debris. 

Citations


View all 10 citations: Citation Explorer
Ventral Stress Fibers Induce Plasma Membrane Deformation in Human Fibroblasts
Authors: Ghilardi, Samuel J and Aronson, Mark S and Sgro, Allyson E
Journal: bioRxiv (2021)
Using the polymeric circulating tumor cell chip to capture circulating tumor cells in blood samples of patients with colorectal cancer
Authors: Kure, Kazumasa and Hosoya, Masaki and Ueyama, Takae and Fukaya, Midori and Sugimoto, Kiichi and Tomiki, Yuichi and Ohnaga, Takashi and Sakamoto, Kazuhiro and Komiyama, Hiromitsu
Journal: Oncology Letters (2020): 2286--2294
The Biological Effects of Interleukin-17A on Adhesion Molecules Expression and Foam Cell Formation in Atherosclerotic Lesions
Authors: Shiotsugu, Shohei and Okinaga, Toshinori and Habu, Manabu and Yoshiga, Daigo and Yoshioka, Izumi and Nishihara, Tatsuji and Ariyoshi, Wataru
Journal: Journal of Interferon & Cytokine Research (2019)
An In Vitro System for Evaluating Molecular Targeted Drugs Using Lung Patient-Derived Tumor Organoids
Authors: Takahashi, Nobuhiko and Hoshi, Hirotaka and Higa, Arisa and Hiyama, Gen and Tamura, Hirosumi and Ogawa, Mayu and Takagi, Kosuke and Goda, Kazuhito and Okabe, Naoyuki and Muto, Satoshi and others, undefined
Journal: Cells (2019): 481
Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-$\kappa$B pathways in human gingival fibroblasts
Authors: Nonaka, Kohei and Kajiura, Yukari and Bando, Mika and Sakamoto, Eijiro and Inagaki, Yuji and Lew, Jung Hwan and Naruishi, Koji and Ikuta, Takahisa and Yoshida, Kaya and Kobayashi, Tetsuo and others,
Journal: Journal of Periodontal Research (2018): 334--344
Addition of granulosa cells collected from differential follicle stages supports development of oocytes derived from porcine early antral follicles
Authors: Ishiguro, Ai and Munakata, Yasuhisa and Shirasuna, Koumei and Kuwayama, Takehito and Iwata, Hisataka
Journal: Reproductive Medicine and Biology (2018)
Autophagy proteins are not universally required for phagosome maturation
Authors: Cemma, Marija and Grinstein, Sergio and Brumell, John H
Journal: Autophagy (2016): 1440--1446
Charakterisierung neuer Inhibitoren des Proteinase aktivierten Rezeptors (PAR) 2 als Ansatz f{\"u}r eine anti-metastatische Tumortherapie
Authors: Stahn, Sonja
Journal: (2016)
Differential detection of tumor cells using a combination of cell rolling, multivalent binding, and multiple antibodies
Authors: Myung, Ja Hye and Gajjar, Khyati A and Chen, Jihua and Molokie, Robert E and Hong, Seungpyo
Journal: Analytical chemistry (2014): 6088--6094
Versatile fabrication of nanoscale sol--gel bioactive glass particles for efficient bone tissue regeneration
Authors: Lei, Bo and Chen, Xiaofeng and Han, Xue and Zhou, Jiaan
Journal: Journal of Materials Chemistry (2012): 16906--16913

References


View all 26 references: Citation Explorer
Requirements, features, and performance of high content screening platforms
Authors: Gough AH, Johnston PA.
Journal: Methods Mol Biol (2007): 41
A pharmaceutical company user's perspective on the potential of high content screening in drug discovery
Authors: Hoffman AF, Garippa RJ.
Journal: Methods Mol Biol (2007): 19
Optimizing the integration of immunoreagents and fluorescent probes for multiplexed high content screening assays
Authors: Giuliano KA., undefined
Journal: Methods Mol Biol (2007): 189
Past, present, and future of high content screening and the field of cellomics
Authors: Taylor DL., undefined
Journal: Methods Mol Biol (2007): 3
High-content fluorescence-based screening for epigenetic modulators
Authors: Martinez ED, Dull AB, Beutler JA, Hager GL.
Journal: Methods Enzymol (2006): 21
Application of laser-scanning fluorescence microplate cytometry in high content screening
Authors: Bowen WP, Wylie PG.
Journal: Assay Drug Dev Technol (2006): 209
High-content screening of known G protein-coupled receptors by arrestin translocation
Authors: Hudson CC, Oakley RH, Sjaastad MD, Loomis CR.
Journal: Methods Enzymol (2006): 63
Evaluation of a high-content screening fluorescence-based assay analyzing the pharmacological modulation of lipid homeostasis in human macrophages
Authors: Werner T, Liebisch G, Gr and l M, Schmitz G.
Journal: Cytometry A (2006): 200
Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay
Authors: Wolff M, Haasen D, Merk S, Kroner M, Maier U, Bordel S, Wiedenmann J, Nienhaus GU, Valler M, Heilker R.
Journal: Comb Chem High Throughput Screen (2006): 339
High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening
Authors: O'Brien P J, Irwin W, Diaz D, Howard-Cofield E, Krejsa CM, Slaughter MR, Gao B, Kaludercic N, Angeline A, Bernardi P, Brain P, Hougham C.
Journal: Arch Toxicol (2006): 580