CD45.2 C57BL/6 (B6) and CD45.1 B6 mice were from the National Cancer Institute or a colony maintained at the University of California, San Francisco. Mice lacking Sphk2 and carrying LoxP-flanked Sphk1 were on a B6/129 mixed background (Pappu et al., 2007 (link)). Lyve-1 Cre knockin mice on a B6/129 mixed background were generated as described in Fig. S1. Lyve-1 Cre+ Sphk1f/− or f/f Sphk2−/− mice were generated by intercrossing. Control mice were usually littermates and were always from the same intercross and carried at least one wild-type Sphk allele. Rosa26-YFP reporter mice (Srinivas et al., 2001 (link)) were provided by N. Killeen (University of California, San Francisco, San Francisco, CA). To generate BM chimeras, recipient CD45.2+ mice were lethally irradiated with 1,300 rads in two doses separated by 3 h and injected with 5 × 106 wild-type BM cells prepared from a CD45.1+ donor. In some experiments, ∼2 × 107 cells/ml were labeled with 3.3 µM CFSE (Invitrogen) or 10 µM 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Invitrogen) in RPMI 1640 containing 2% FCS for 20 min at 37°C, and were then washed by spinning through a layer of FCS. Labeled cells were resuspended at ∼2 × 107 cells/ml, and were treated with 10 ng/ml OB or PTX at 37°C for 10 min, washed twice in warm RPMI 1640 with 2% FCS and 10 mM Hepes, and transferred to recipient mice. Lymph collection was performed as previously described (Matloubian et al., 2004 (link)). In brief, under a stereomicroscope, lymph was drawn from the cysterna chyli using a fine borosilicate glass microcapillary pipette (Sutter Instrument Co.). Cell numbers determined by flow cytometry were divided by the volume of collected lymph to determine the concentration. Protocols were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.
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(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
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Most cited protocols related to «(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine»
(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
5-(6)-carboxyfluorescein diacetate succinimidyl ester
Alleles
Cells
Chimera
Flow Cytometry
HEPES
Institutional Animal Care and Use Committees
Lymph
Mice, 129 Strain
Mus
RRAD protein, human
sphingosine kinase 2, human
tetramethylrhodamine
Tissue Donors
(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
Axilla
Cells
Groin
IL2RA protein, human
Mus
Nodes, Lymph
Population Group
Regulatory T-Lymphocytes
Reperfusion
Tail
Therapeutic Effect
Veins
Spheroids made of 3T3 cells were collected from U-bottom 96-well plates and then washed with DPBS twice. CellTracker Orange CMTMR [5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine] (Thermo Fisher Scientific) and CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) (Thermo Fisher Scientific, MA) were used according to the manufacturer’s instructions. Spheroids were bioprinted with close proximity, and then, images were captured at 0, 24, 48, and 72 hours using EVOS FL Cell Imaging System. The contact length and contact angle of two spheroids were measured every 4 hours up to 24 hours.
(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
3T3 Cells
5-chloromethylfluorescein diacetate
Cells
tetramethylrhodamine
For the assay, wound macrophages were seeded in 8-well chambered slides. Apoptotic (5 µM dexamethasone treated for 12 h; yield >90% PS positive thymocytes, Fig. 3A ) thymocytes were added to each chamber in a (1∶10) macrophage:thymocyte ratio. Prior to co-culture with macrophages, thymocytes were labeled with a fluorescence cell-tracker reagent (CellTracker™ Orange CMTMR, Molecular Probes). Thymocytes have been largely used and are well accepted for efferocytosis studies performed using cultured macrophage ex vivo. Moreover, upon induction of apoptosis, both PMN and thymocytes are known to externalize phosphatidyl serine (PS), one of the key mechanisms of apoptotic cell recognition by macrophages [74] (link), [75] (link). Phagocytosis assay was performed for 1 h at 37°C. In co-culture studies, shorter incubation times (10–15 min) were used for adherence assay while longer (45–60 min) co-culture period were utilized for the phagocytosis assays [51] (link). Macrophages were then extensively washed to remove non-phagocytosed cells. Cells were fixed with 4% paraformaldehyde and stained using F4/80-FITC. Imaging was performed using a fluorescence microscope (thymocytes, red; macrophage green or phase contrast). Quantitation of phagocytosed thymocytes by each macrophage was performed using Axiovision software (Zeiss) by counting 50–100 macrophages from each well. Data are expressed as “phagocytic index”. This index is defined as the total number of apoptotic cells engulfed per macrophage present in the field of view [62] (link). This approach enables normalization of the data against macrophage number.
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(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
Apoptosis
Biological Assay
Cells
Coculture Techniques
Dexamethasone
Fluorescein-5-isothiocyanate
Fluorescence
Macrophage
Microscopy, Fluorescence
Microscopy, Phase-Contrast
Molecular Probes
paraform
Phagocytes
Phosphatidylserines
Thymocyte
Wounds
BM cells from femurs of CCR7+/+ or CCR7−/− mice were cultured in RPMI 1640 (GIBCO BRL) supplemented with 10% FCS (HyClone) and recombinant mouse GM-CSF (R&D Systems) according to current protocols. To induce maturation, 8–10 d BM-DC cultures were stimulated with 0.5 μg/ml LPS (Sigma-Aldrich) for 24 h. At that time point DCs were homogeneously CCR7+ as detected by staining with a CCL21–Ig fusion protein (provided by K. Karjalainen, Institute for Research in Biomedicine, Bellinzona, Switzerland). CD11c+ and CD11c− spleen cells were isolated using CD11c-coated magnetic beads (Miltenyi Biotec). Cells were labeled with 2.5 μM 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) or 10 μM 5- and 6-(4-chloromethyl)benzoyl-amino-tetramethylrhodamine (CMTMR) intracellular fluorescent dyes according to the manufacturer's instructions (Molecular Probes). After labeling, cells were washed extensively in PBS and injected s.c. into the footpad or flank (for experiments involving use of CFA or IFA). No differences in the extent of DC migration to lymph nodes were found when CFSE or CMTMR was used.
(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
5-(6)-carboxyfluorescein diacetate succinimidyl ester
CCL21 protein, human
Cells
Femur
Fluorescent Dyes
Granulocyte-Macrophage Colony-Stimulating Factor
Molecular Probes
Mus
Nodes, Lymph
Proteins
Protoplasm
Spleen
tetramethylrhodamine
Most recents protocols related to «(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine»
The HT29 and DLD-1 cells were labeled with CellTracker Orange CMTMR Dye C2927 (Thermo Fisher Scientific), and the cell suspension was adjusted to 500 cells per 50 μL by serial dilution. The actual number of cells in 10 μL cell suspension was directly counted under a fluorescence microscope (BZ-X800). Next, a cell suspension, containing approximately 500–700 cells, was mixed with 10 mL of venous peripheral blood from a healthy donor, and added to an OncoQuick tube (Greiner bio-one, Kremsmünster, Austria). After centrifugation at 1600× g for 20 min at 4 °C, the cells were washed once with PBS, washed again with advanced DMEM (Thermo Fisher Scientific), and then collected in 1 mL standard medium.
One 200-μL-aliquot of the 1 mL cell suspension was used to count the CRC cells marked with orange fluorescence under a fluorescence microscope. Another 200-μL-aliquot was transferred into a chamber slide II (IWAKI) coated with 0.25% (w/v) PMEA in methanol solution and 200 μg/mL fibronectin (in duplicate). These cells were incubated in standard medium at 37 °C overnight. The next day, immunocytochemistry for EpCAM was performed, and stained tumor cells were counted under a microscope in the light field.
One 200-μL-aliquot of the 1 mL cell suspension was used to count the CRC cells marked with orange fluorescence under a fluorescence microscope. Another 200-μL-aliquot was transferred into a chamber slide II (IWAKI) coated with 0.25% (w/v) PMEA in methanol solution and 200 μg/mL fibronectin (in duplicate). These cells were incubated in standard medium at 37 °C overnight. The next day, immunocytochemistry for EpCAM was performed, and stained tumor cells were counted under a microscope in the light field.
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(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
BLOOD
Cells
Centrifugation
Fibronectins
Fluorescence
HT29 Cells
Immunocytochemistry
Light
Methanol
Microscopy
Microscopy, Fluorescence
Neoplasms
TACSTD1 protein, human
Technique, Dilution
Tissue Donors
Veins
The CRC cell lines HCT116, SW480, and HT29 were labeled with CellTracker Orange CMTMR Dye C2927 (Thermo Fisher Scientific, Waltham, MA, USA). Cell suspensions were adjusted to 100 cells per 10 μL by serial dilution, and the actual number of cells in 10 μL cell suspension was directly counted under a fluorescence microscope BZ-X800 (KEYENCE, Osaka, Japan). With orange fluorescence as an indicator, 100 cells were seeded (in triplicate) on 12-well tissue-culture-treated polystyrene (TCPS) dishes (IWAKI, Shizuoka, Japan), or on dishes coated with 0.25% (w/v) PMEA in methanol solution and 200 μg/mL fibronectin. Cells were incubated in standard medium at 37 °C overnight, and then washed twice with PBS. The attached cells were counted under a fluorescence microscope (BZ-X800).
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(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
Cell Lines
Cells
Fibronectins
Fluorescence
HT29 Cells
Hyperostosis, Diffuse Idiopathic Skeletal
Methanol
Microscopy, Fluorescence
Polystyrenes
Technique, Dilution
Tissues
CTLs were transferred from T75 flasks into 15 mL Falcon tubes, and the cell number was adjusted to either 1 × 105 cytotoxic T cells (spheroid coculture) or 1 × 106 CTLs (DSFC) and stained for 45 min at 37 °C with 1 µM CellTracker™ Orange CMTMR (Invitrogen, stock solution 1 mM) in PBS. CellTracker™ Orange CMTMR is nontoxic and, after passing through a cell’s outer cell membrane, is converted into a reaction product that cannot penetrate the cell membrane. CellTracker dyes transfer to daughter cells, but do not transfer to adjacent cells. After staining, CTLs were resuspended in T-cell media and added to spheroids or resuspended in 50 µL PBS for intraperitoneal injection into tumor-bearing mice with DSFC.
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(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
Cells
Coculture Techniques
Cytotoxic T-Lymphocytes
Daughter
Dyes
Injections, Intraperitoneal
Mus
Neoplasms
Plasma Membrane
T-Lymphocyte
FACS-sorted cells were washed with DPBS and quantified. Cardiac macrophages (2 × 105 cells) were resuspended in 25 μl MG (Corning). After anesthetizing and intubating the recipient mice, the mouse chest was opened and MG containing cardiac macrophages was injected into the pericardial cavity using a 30-gauge needle. The control animals were only intrapericardially injected with 25 μl MG.
For cell survival and distribution experiments, the sorted Mac1 and non-Mac1 cells were stained with CMTMR (50 nM) at 37 °C for 20 min and then washed with DPBS. Labeled macrophages (2 × 105 cells) were resuspended in 25 μl MG and then transplanted into the pericardial cavity immediately after CLP. Three days later the mice were killed. Heart samples were collected and cryosections were made. Images were acquired using a Zeiss LSM 880 (with fast AiryScan) according to standard procedures and analyzed with ImageJ Pro Plus v.6.0 software.
For cell survival and distribution experiments, the sorted Mac1 and non-Mac1 cells were stained with CMTMR (50 nM) at 37 °C for 20 min and then washed with DPBS. Labeled macrophages (2 × 105 cells) were resuspended in 25 μl MG and then transplanted into the pericardial cavity immediately after CLP. Three days later the mice were killed. Heart samples were collected and cryosections were made. Images were acquired using a Zeiss LSM 880 (with fast AiryScan) according to standard procedures and analyzed with ImageJ Pro Plus v.6.0 software.
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(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
Animals
Cells
Cell Survival
Chest
Cryoultramicrotomy
Heart
Macrophage
Mus
Needles
Pericardial Cavity
MSCs stained with CellTracker Orange CMTMR were either prepared in suspension or loaded in 35 µm nanovials. This stain was chosen to avoid spectral overlap with later live/dead Incucyte imaging. For MSCs in suspension, FACS buffer (PBS without calcium and magnesium + 1% A-A + 0.5% BSA) or FACS buffer with 0.05% Pluronic was used. For MSCs in nanovials, wash buffer (PBS with calcium and magnesium + 1% A-A + 0.5% BSA + 0.05% Pluronic) or wash buffer without 0.05% Pluronic was used. Immediately after straining the sample as described previously, samples were sorted. The 405 nm, 488 nm, and 561 nm lasers were turned on for this experiment. Samples were sorted per well in a 96 black well plate in triplicate, and a similar amount of unsorted sample was prepared. Then calcein AM and propidium iodide were added to the existing media plus sorted sample resulting in a final 1:1000 dilution of each stain, and incubated for 30 minutes before imaging on the IncuCyte Live-Cell Analysis System using phase, green and red channels. Fluorescent images were thresholded manually, and the number of live/dead cells were quantified using the IncuCyte S3 software.
(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine
Buffers
Calcium
Cells
fluorexon
Magnesium
Pluronics
Propidium Iodide
Stains
Technique, Dilution
Top products related to «(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine»
Sourced in United States, United Kingdom
CellTracker Orange CMTMR is a fluorescent cell-permeant dye used to label living cells. It can be used for tracking and visualizing cells in various applications such as cell biology and developmental biology.
CMTMR is a fluorogenic cell-permeant dye that can be used to track and quantify cell populations. It is a red-orange fluorescent indicator that can be used to measure cell viability and proliferation.
Sourced in United States
CellTracker Orange CMTMR Dye is a fluorescent cell-permeant dye used for labeling and tracking cells. It is useful for visualizing and monitoring cell populations, particularly in live-cell imaging applications.
Sourced in United States, Germany, United Kingdom, Canada, France, China, Australia, Belgium, Denmark, Netherlands, Norway, Italy, Sweden, New Zealand
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye used for cell proliferation and tracking assays. It binds to cellular proteins, allowing the labeling and monitoring of cell division in various cell types.
Sourced in United States, United Kingdom, Germany, Belgium, Japan, Canada
CellTracker Green CMFDA is a fluorescent dye that can be used to stain live cells. It is designed to passively diffuse into cells and become fluorescently labeled, allowing for the visualization and tracking of cell populations.
CellTracker CMTMR is a fluorescent dye used for long-term cell labeling. It is a cell-permeant dye that freely diffuses into cells and gets converted to a cell-impermeant form, allowing for long-term labeling and tracking of cells.
Sourced in United States, United Kingdom
CellTracker Orange is a fluorescent dye that can be used to label and track living cells. It is a cell-permeant dye that becomes fluorescent upon entering the cell and remains there, allowing for the visualization and monitoring of cellular processes.
Sourced in United States
CellTracker orange CMTMR is a fluorescent dye used to label live cells. It is a cell-permeant dye that becomes fluorescent upon entering the cell and reacting with intracellular thiols, enabling the visualization and tracking of labeled cells.
Sourced in United States
The CMTMR Dye is a fluorescent dye that can be used to label and track cells in various biological applications. It is a cell-permeant dye that emits fluorescence upon binding to thiol groups within the cells. The dye can be used to monitor cell viability, proliferation, and trafficking.
Orange CMTMR is a fluorescent dye used in cell biology applications. It is a derivative of the cell-permeant dye 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR). The dye can be used to label and track cells by covalently binding to intracellular components.
More about "(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine"
(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine is a fluorescent dye used for cell tracing and labeling in biological research.
It is a derivative of tetramethylrhodamine, a popular fluorescent marker with excitation and emission wavelengths suitable for many microscopy and flow cytometry applications. (((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine, also known as CellTracker Orange CMTMR or simply CMTMR, is frequently used to label live cells and track their migration, proliferation, and fate in various experimental models.
The chloromethyl group in the molecule allows the dye to passively diffuse into cells and become covalently bound to intracellular components, ensuring its retention within the labeled cells.
This makes CMTMR a useful tool for long-term cell tracking studies.
CMTMR has spectral properties similar to other rhodamine-based dyes like CFSE and CellTracker Green CMFDA, allowing multicolor labeling and analysis.
In addition to its use in cell tracing, CMTMR has been employed in a variety of other applications, such as measuring cell viability, apoptosis, and phagocytosis.
Reseaerchers can utilize PubCompare.ai to discover optimized, reproducible protocols for working with (((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine, drawing from the published literature, preprints, and patents.
This can help improve research efficiency and obtain better results when using this versitile fluorescent dye.
It is a derivative of tetramethylrhodamine, a popular fluorescent marker with excitation and emission wavelengths suitable for many microscopy and flow cytometry applications. (((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine, also known as CellTracker Orange CMTMR or simply CMTMR, is frequently used to label live cells and track their migration, proliferation, and fate in various experimental models.
The chloromethyl group in the molecule allows the dye to passively diffuse into cells and become covalently bound to intracellular components, ensuring its retention within the labeled cells.
This makes CMTMR a useful tool for long-term cell tracking studies.
CMTMR has spectral properties similar to other rhodamine-based dyes like CFSE and CellTracker Green CMFDA, allowing multicolor labeling and analysis.
In addition to its use in cell tracing, CMTMR has been employed in a variety of other applications, such as measuring cell viability, apoptosis, and phagocytosis.
Reseaerchers can utilize PubCompare.ai to discover optimized, reproducible protocols for working with (((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine, drawing from the published literature, preprints, and patents.
This can help improve research efficiency and obtain better results when using this versitile fluorescent dye.