Fluorescent dye
CellTracker Blue CMAC targets cellular proteins through its chloromethyl group, which forms covalent bonds with thiol groups on proteins. Once inside cells, the compound is converted to a fluorescent, glutathione-conjugated product that is well retained and passed to daughter cells while remaining non-toxic. The dye does not have a specific biological receptor target but is used as a cell labeling tool for tracking cell movement, migration, and proliferation.

CellTrack Blue CMAC (Known as CellTracker™ Blue CMAC, Trade Mark of Molecular Probes) is a fluorescent thiol-reactive cell permeant probe that easily pass through cell membranes. Upon reacting with thio-containing compound, the probe is well retained in cells and is passed to daughter cells through several generations but is not transferred to adjacent cells in the population. Cells that are loaded with this probe are typically fluorescent and viable for at least 24 hours, making these probes excellent long-term cell tracers. Furthermore, this probe can be fixed with formaldehyde or glutaraldehyde to allow long-term storage of the labeled cells or tissue, to facilitate safe handling of potentially pathogenic samples.

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Advantages of CellTracker Blue CMAC:
1) Easy to use - remove culture medium, add dye, incubate for 30 minutes, perform cell imaging
2) Fluorescence signal retention>72 hours (usually 3 to 6 generations)
3) Blue excitation/emission spectrum suitable for multi-channel technology (maximum value of 353/466 nm)
4) Low cytotoxicity - does not affect viability or proliferation

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CellTracker Blue CMAC is a fluorescent dye used for long-term labeling and tracking of live cells. The dye's chloromethyl group forms covalent bonds with cellular proteins, providing stable attachment. Once inside cells, it is converted to a fluorescent, glutathione-conjugated product that is well retained and passed to daughter cells. The dye emits bright blue fluorescence, enabling visualization of cell movement. It is commonly used in studies of cell migration, cancer research, immune response, and stem cell tracking.

An inclusive chemical definition of “reactive” dyeing of textiles is introduced, encompassing the CI Azoic, CI Mordant, CI Reactive, CI Sulphur and CI Vat dye application classes. Such reactive dyeing increases fibre retention of dye and makes application practically possible. The analogous application of dyes and fluorescent probes as microscopic stains in biology and medicine is outlined, focussing on using reactive fluorescent probes with living cells. Parallels with textile dyeing are noted, eg, enhanced probe retention and facilitation of probe application. However, the primary purpose of using reactive probes with live cells is detection of properties of biological systems: to identify biological structures and chemical/biochemical contents; assess biological functions and physicochemical properties; and determine changes in locations of cells and cell components. Problems occurring with such probes are outlined, particularly the problematic character of many standard protocols, and localisation artefacts arising with reactive probes whose reactant and product species are physiochemically significantly different. This latter problem is explored via a case study of possible reactant/product artefacts with probes for reactive oxygen species. Comparison of experimental observations of probe localisations with the localisations predicted using quantitative structure activity (QSAR) modelling indicates that such artefacts can occur with a significant proportion of chemically diverse, widely used, commercially available probes, as well as with experimental compounds reported in the literature. A graphical flowchart is provided to assess possible occurrence of reactant/product artefacts arising with reactive fluorescent probes localising in various organelles of living cells [1].

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In vivo, CellTracker Blue CMAC is used for cell tracking in live organisms. The dye's low toxicity and stable retention make it suitable for long-term cell tracking studies. It can be used to label cells prior to transplantation or injection into animal models to study cell migration, homing, and engraftment. The dye's blue fluorescence allows for visualization of labeled cells in tissues. Specific in vivo efficacy studies are not applicable, as the dye is used as a cell labeling tool rather than a therapeutic agent.

CellTracker Blue CMAC Dye is a fluorescent dye that is very suitable for monitoring cell movement or position. CellTracker Blue CMAC fluorescent dye can freely pass through the cell membrane and enter the cell, and is converted into a reactive product that does not penetrate the cell membrane within the cell. This dye can be transferred to daughter cells, but it will not transfer to adjacent cells in the population. The CellTracker Blue CMAC dye is designed to display fluorescence for at least 72 hours and has ideal tracking properties: stable at working concentrations, non-toxic, well retained in cells, and emitting bright fluorescence at physiological pH values. In addition, The excitation and emission spectra of CellTracker Blue CMAC dye are completely separated from GFP (green fluorescent protein) and RFP (red fluorescent protein) spectra and can be multiplexed.

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In vitro labeling assays for CellTracker Blue CMAC typically involve incubating cells with the dye at concentrations of 1-25 μM for 15-45 minutes at 37°C. The dye is dissolved in DMSO as a stock solution and diluted in serum-free or low-serum culture medium. Following incubation, cells are washed to remove unbound dye and then cultured in complete medium. Labeled cells are analyzed by fluorescence microscopy using blue fluorescence excitation/emission filters or by flow cytometry. The dye's retention and stability are assessed over time.

Experimental procedure:
1. Dissolve the CMAC solid in anhydrous DMSO and prepare a 10 mM stock solution. Dilute the stock solution to a working solution of 0.5-25 μ M using serum-free culture medium. Preheat the working fluid at 37 ℃ before use
2. Incubate for 15-45 minutes under different cell culture conditions according to different cell types. For suspended cells, centrifuge the supernatant and collect cell precipitates. Resuspend cells with preheated staining solution; For adherent cells, when the cell grows to the appropriate cell density, remove the supernatant culture medium and add preheated staining solution
3. Remove the staining solution, add fresh preheated cell culture medium, and incubate at 37 ℃ for 30 minutes
4. For suspended cells, draw about 5 μ l of stained cells and drop them onto a glass slide. Cover the slide with a cover glass and observe under a fluorescence microscope; For adherent cells, direct observation is sufficient. (Optional) If you need to fix the cells, you can skip step 4 first and prepare for observation after the fixation step is completed
5. Use PBS to wash cells
6. Fix cells with PBS containing 3.7% paraformaldehyde and incubate at room temperature for 15 minutes
7. Use PBS to wash cells
8. If necessary, the cell membrane can be permeabilized. When cells need to be labeled with antibodies, membrane permeabilization is necessary to facilitate antigen entry into the cells. Cold acetone can be used to treat cells for 10 minutes, which can provide cell permeability.

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In vitro cellular assays for CellTracker Blue CMAC typically involve labeling cells for tracking and proliferation studies. Cells are incubated with the dye at concentrations of 1-25 μM for 15-45 minutes at 37°C. Labeled cells are then cultured and monitored over time using fluorescence microscopy or flow cytometry. Cell proliferation is assessed by the dilution of fluorescence as cells divide. Cell migration is assessed using Transwell or wound healing assays. The dye's non-toxic nature allows for long-term tracking.

In vivo animal studies for CellTracker Blue CMAC typically involve labeling cells with the dye prior to transplantation into animal models. Labeled cells are administered via injection (intravenous, subcutaneous, or local), and the cells are tracked over time by fluorescence imaging of tissues. The dye's stable retention allows for long-term tracking of cell migration, homing, and engraftment. Tissue sections are examined by fluorescence microscopy. The dye's low toxicity makes it suitable for in vivo applications.

CellTracker Blue CMAC has a molecular weight of 209.63 g/mol and formula C10H8ClNO2. It is a cell-permeant dye that is soluble in DMSO. Recommended storage is at -20°C, protected from light. Purity is ≥98%. The dye is typically supplied as a powder and should be stored desiccated. Detailed PK parameters are not applicable for this cell labeling dye, as it is not used as a therapeutic agent.

CellTracker Blue CMAC has low toxicity to cells, making it suitable for long-term live cell tracking. The compound is intended for research use only and is not approved for human therapeutic applications. As with all chemical compounds, appropriate safety precautions should be taken when handling, including wearing gloves and protective eyewear. The dye should be protected from light to prevent photodegradation.

[1]. Reactive dyes for living cells: Applications, artefacts, and some comparisons with textile dyeing. Coloration technology, 2022,138(1):3-15.

CellTracker Blue CMAC (CAS 147963-22-2) is a cell-permeant, thiol-reactive blue fluorescent dye used for long-term labeling and tracking of live cells. It has a molecular weight of 209.63 g/mol and formula C10H8ClNO2. The dye's chloromethyl group forms covalent bonds with cellular proteins, providing stable attachment. It is commonly used in studies of cell migration, cancer research, immune response, and stem cell tracking. The compound is also known as 7-amino-4-chloromethylcoumarin. It is not approved for clinical use.

C10H8NO2CL

209.62902

209.024

147963-22-2

2762598

White to off-white solid powder

1.399 g/cm3

410.3ºC at 760 mmHg

201.9ºC

6.1E-07mmHg at 25°C

1.633

2.695

1

3

1

14

277

0

C1=CC2=C(C=C1N)OC(=O)C=C2CCl

VIEYMVWPECAOCY-UHFFFAOYSA-N

InChI=1S/C10H8ClNO2/c11-5-6-3-10(13)14-9-4-7(12)1-2-8(6)9/h1-4H,5,12H2

7-amino-4-(chloromethyl)chromen-2-one; 7-AMINO-4-CHLOROMETHYLCOUMARIN

7-amino-4-chloromethylcoumarin; 7-amino-4-(chloromethyl)-2H-chromen-2-one; 7-amino-4-(chloromethyl)chromen-2-one; CellTracker Blue CMAC; 147963-22-2; 7-amino-4-(chloromethyl)-2H-chromen-2-one; 7-amino-4-(chloromethyl)chromen-2-one; 2H-1-Benzopyran-2-one, 7-amino-4-(chloromethyl)-; CMAC cpd; CMAC; .

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~596.29 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (9.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)