CellTracker fluorescent probes Product Information Sheet (Pub. No. MAN0001826 B.0)
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CellTracker fluorescent probes Product Information Sheet (Pub. No. MAN0001826 B.0)
Product description CellTracker fluorescent probes
Figure 1 CellTracker™ fluorescence probes display no cytotoxicity. CellTracker™ Blue (CTB), CellTracker™ Green (CTG), CellTracker™ Red (CTR), and CellTracker™ Deep Red (CTDR) were loaded into HeLa cells following manufa…
[PDF] CellTracker fluorescent probes Product Information Sheet (Pub. No ...
Dec 10, 2018 · CellTracker™ Blue (CTB), CellTracker™ Green (CTG), CellTracker™ Red. (CTR), and CellTracker™ Deep Red (CTDR) were loaded into HeLa cells ...
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PRODUCT INFORMATION SHEET CellTrackerTM fluorescent probes Catalog Numbers C2102, C2110, C2111, C2925, C2927, C7025, C10094, C12881, C34551, C34552, and C34565 Pub. No. MAN0001826 Rev. B.0 Contents and storage Material Amount CellTrackerTM Blue dyes and CellTrackerTM Green BODIPYTM 5 mg dry powder dyes CellTrackerTM Violet BMQC dye 5 � 0.1 mg dry powder CellTrackerTM Green CMFDA and CellTrackerTM Orange CMTMR dyes 1 mg dry powder CellTrackerTM Green CMFDA, CellTrackerTM Orange CMRA, 20 � 50 �g dry powder and CellTrackerTM Red CMTPX dyes CellTrackerTM Deep Red dye 20 � 15 �g dry powder Approximate fluorescence excitation/emission maxima: See Table 1. Storage � �20�C � Desiccate � Protect from light Stability The product is stable for 1 year when stored as directed Product description Cell movement and location studies require specialized probes that are nontoxic to living cells and are available in a range of fluorescent colors to match instrument lasers and filters and to accommodate co-staining with antibodies or other cell analysis probes. The CellTrackerTM fluorescent probes are excellent tools for monitoring cell movement, location, proliferation, migration, chemotaxis, and invasion. The CellTrackerTM fluorescent probes have been designed to freely pass through cell membranes; however, once inside the cell are transformed into cell-impermeant reaction products. The CellTrackerTM fluorescence probes (except for CellTrackerTM Deep Red) contain a chloromethyl or bromomethyl group that reacts with thiol groups, utilizing a glutathione S-transferase�mediated reaction. In most cells, glutathione levels are high (up to 10 mM) and glutathione transferase is ubiquitous. CellTrackerTM Deep Red probe contains a succinimidyl ester reactive group, which reacts with amine groups present on proteins. After conversion to impermenant versions, the CellTrackerTM fluorescent probes are well retained in living cells through several generations. The probes are transferred to daughter cells, but are not transferred to adjacent cells in a population. Cells loaded with the CellTrackerTM fluorescent probes display fluorescence for at least 72 hours and exhibit ideal tracking dye properties--they are stable, nontoxic at working concentrations (Figure 1), well retained in cells, and brightly fluorescent at physiological pH. Additionally, several CellTrackerTM fluorescent probes with various excitation and emission spectra are available (Table 1) allowing for multiplexing. For Research Use Only. Not for use in diagnostic procedures. Figure 1 CellTrackerTM fluorescence probes display no cytotoxicity. CellTrackerTM Blue (CTB), CellTrackerTM Green (CTG), CellTrackerTM Red (CTR), and CellTrackerTM Deep Red (CTDR) were loaded into HeLa cells following manufacture instructions. Time points of day 1, 2, and 3 post-loaded were harvested and proliferation was measured using the CyQUANTTM direct cell proliferation assay. RFU = Relative fluorescence units. Table 1 Spectral characteristics of the fluorescent CellTrackerTM probes. Cat. No. CellTrackerTM Probe MW Ex [1] (nm) Em [1] (nm) C2110 CellTrackerTM Blue CMAC (7-amino-4chloromethylcoumarin) 209.6 353 466 C12881 CellTrackerTM Blue CMF2HC (4-chloromethyl-6,8- 246.6 371 464 difluoro-7-hydroxycoumarin) C2111 CellTrackerTM Blue CMHC (4-chloromethyl-7hydroxycoumarin) 210.6 372 470 C10094 C2925, C7025 CellTrackerTM Violet BMQC (2,3,6,7-tetrahydro-9bromomethyl-1H,5H-quinolizino(9,1gh)coumarin) CellTrackerTM Green CMFDA (5chloromethylfluorescein diacetate) 334.2 464.9 415 492 [2] 516 517 [2] C2102 CellTrackerTM Green BODIPYTM (8-chloromethyl-4,4- 296.6 522 529 difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s- indacene) C2927 CellTrackerTM Orange CMTMR (5-(and-6)-(((4- 554.0 541 565 chloromethyl)benzoyl)amino) tetramethylrhodamine C34551 CellTrackerTM Orange CMRA 550.4 548 576 C34552 CellTrackerTM Red CMTPX 686.3 577 602 C34565 CellTrackerTM Deep Red 698.3 630 660 [1] Absorption and fluorescence emission maxima, determined in aqueous buffer or methanol; values may vary somewhat in cellular environments. [2] CMFDA is colorless and nonfluorescent until the acetate groups are cleaved by intracellular esterases; hydrolysis of the acetates yields a product with the indicated spectral properties. MW = Molecular weight. Fluorescent CellTrackerTM reagents include the blue-fluorescent chloromethyl derivatives of amino-, hydroxy-, and difluorohydroxycoumarin (CMAC, CMHC and CMF2HC), the green fluorescent chloromethyl derivatives of fluorescein diacetate (CMFDA) and a BODIPYTM dye, the orange-fluorescent CMTMR and CMRA, and the red-fluorescent CMTPX. CellTrackerTM Blue CMAC, CMHC, and CMF2HC, CellTrackerTM Violet, the violet-fluorescent bromomethyl derivative of coumarin (BMQC), CellTrackerTM Green BODIPYTM, CellTrackerTM Orange CMTMR, and CellTrackerTM Red CMTPX do not require enzymatic cleavage to activate their fluorescence, whereas the green CMFDA and orange CMRA do require enzymatic cleavage. The impermeable reaction products of the chloromethyl or bromomethyl coumarins have excellent retention, strong fluorescence, and relatively uniform cytoplasmic staining, making these derivatives potentially useful for correcting motion artifacts in imaging. CMFDA is colorless and non-fluorescent until cytosolic esterases cleave off the acetates, releasing a brightly fluorescent product. In addition to bright fluorescence and excellent retention the CellTrackerTM fluorescent probes do not contribute to cytotoxicity (Figure 1). 2 CellTrackerTM fluorescent probes Product Information Sheet Before starting CAUTION! No data are available addressing the mutagenicity or toxicity of CellTrackerTM reagents (Component A). Handle the DMSO dye solution with caution because DMSO is known to facilitate the entry of organic molecules into tissues. Always wear suitable protective clothing, gloves, and eye/face protection when handling this reagent. Dispose of the reagents in compliance with all pertaining local regulations. IMPORTANT! Avoid amine- and thiol-containing buffers. Materials required but not provided � Anhydrous dimethylsulfoxide (DMSO) � Phosphate-buffered saline (PBS) Prepare cells Grow cells in an appropriate culture medium. Adherent cells can be grown on coverslips inside a petri dish filled with culture medium. Experimental protocols The following protocol describes introducing the reagent into cultured cells and imaging the stained cells by fluorescence microscopy. Various factors, such as penetration of the dye into the cells or tissue, may require that some conditions be modified for particular cell types. The optimal concentration of the probe for staining varies depending upon the application. We recommend testing at least a ten-fold range of concentrations. In general, long-term staining (more than about 3 days) or the use of rapidly dividing cells requires 5�25 �M dye. Less dye (0.5�5 �M) is needed for shorter experiments, such as viability assays. Due to the high fluorescent signal resulting from staining with Deep Red dye, the optimal concentration for this dye is 250 nM�1 �M. To maintain normal cellular physiology and reduce potential artifacts, keep the dye concentration as low as possible. The effects of overloading may not be immediately apparent. For example, peripheral blood lymphocytes respond normally to concanavalin A when treated with up to 1 �M dye, but not with more than 5 �M dye. Prepare working dye solution Before opening the dye vial, allow the product to warm to room temperature. Dissolve the lyophilized product in high-quality DMSO to a final concentration of 10 mM. For CellTrackerTM Deep Red dye, add 20 �L of DMSO per vial to make a 1 mM (1000X) solution. Dilute the stock solution to a final working concentration of 0.5�25 �M in serumfree medium. Warm the CellTrackerTM Working Solution to 37�C. Note: For the molecular weight of CellTrackerTM reagents, see Table 1. Staining protocol for cells in suspension 1. Harvest cells by centrifugation and aspirate the supernatant. Resuspend the cells gently in pre-warmed CellTrackerTM Working Solution (see "Prepare working dye solution"). 2. Incubate 15�45 minutes under growth conditions appropriate for the particular cell type. 3. Centrifuge the cells and remove the CellTrackerTM Working Solution. 4. Add culture media of choice and dispense the labeled cell onto slide or into a culture vessel of choice. 5. Image using the appropriate emission and excitation filters for the CellTrackerTM probe (Table 1). Staining protocol for adherent cells 1. Remove culture media. 2. Gently add the pre-warmed CellTrackerTM Working Solution (see "Prepare working dye solution" ). 3. Incubate 15�45 minutes under growth conditions appropriate for the particular cell type. 4. Remove the CellTrackerTM Working Solution. 5. Add culture media of choice. 6. Image using the appropriate emission and excitation filters for the CellTrackerTM probe (Table 1). Fluorescence microscopy The CellTrackerTM probes can be used on a wide range of epifluorescence microscopes with standard optics and video enhancement. Select optical filters according to the dye. Table 1 summarizes the spectral characteristics of the CellTrackerTM probes. References 1. FASEB J 6, A1835 (1992); 2. Nature 363, 549 (1993); 3. Cytometry 14, 747 (1993); 4. Cytometry 12, 184 (1991); 5. Toxicol Appl Pharmacol 112, 235 (1992); 6. Anal Biochem 216, 271 (1994); 7. Biophys J 67, 1574 (1994). CellTrackerTM fluorescent probes Product Information Sheet 3 Product list Ordering information for products available from Thermo Fisher Scientific is provided below. For detailed information, visit our website at thermofisher.com or call Technical Support. C2110 C12881 C2111 C10094 C2102 C2925 C7025 C34551 C2927 C34552 C34565 Cat. No. Product Name CellTrackerTM Blue CMAC (7-amino-4-chloromethylcoumarin) CellTrackerTM Blue CMF2HC (4-chloromethyl-6,8-difluoro-7hydroxycoumarin) CellTrackerTM Blue CMHC (4-chloromethyl-7-hydroxycoumarin) CellTrackerTM Violet BMQC (2,3,6,7-tetrahydro-9- bromomethyl-1H,5Hquinolizino(9,1gh)coumarin) CellTrackerTM Green BODIPYTM (8-chloromethyl-4,4-difluoro-1,3,5,7tetramethyl-4-bora-3a,4a-diaza-s-indacene) CellTrackerTM Green CMFDA (5-chloromethylfluorescein diacetate) CellTrackerTM Green CMFDA (5-chloromethylfluorescein diacetate) *special packaging* CellTrackerTM Orange CMRA *special packaging* CellTrackerTM Orange CMTMR (5-(and-6)-(((4chloromethyl)benzoyl)amino)tetramethylrhodamine) *mixed isomers* CellTrackerTM Red CMTPX *special packaging* CellTrackerTM Deep Red Unit Size 5 mg 5 mg 5 mg 5 � 100 �g 5 mg 1 mg 20 � 50 �g 20 � 50 �g 1 mg 20 � 50 �g 20 � 15 �g Limited product warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support. Life Technologies Corporation | 29851 Willow Creek | Eugene, OR 97402 For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition. The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history: Pub. No. MAN0001826 Revision B.0 Date Nov 2018 Baseline for publication. Description Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. �2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. thermofisher.com/support | thermofisher.com/askaquestion thermofisher.com 10 December 2018