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Carboxyfluorescein
Carboxyfluorescein
Carboxyfluorescein is a fluorescent dye commonly used as a tracer in biological and medical research.
It is a derivative of fluorescein, with a carboxyl group attached to the molecule.
Carboxyfluorescein is often used to label cells, proteins, or other biomolecules, allowing their visualization and tracking in various experimental settings.
It has excitation and emission wavelengths that make it compatible with common fluorescence detection equipment.
Carboxyfluorescein is also used in assays to measure cell viability, proliferation, and other cellular processes.
Its versatile properties and ease of use have made it a valuable tool in the fields of cell biology, immunology, and drug discovery.
It is a derivative of fluorescein, with a carboxyl group attached to the molecule.
Carboxyfluorescein is often used to label cells, proteins, or other biomolecules, allowing their visualization and tracking in various experimental settings.
It has excitation and emission wavelengths that make it compatible with common fluorescence detection equipment.
Carboxyfluorescein is also used in assays to measure cell viability, proliferation, and other cellular processes.
Its versatile properties and ease of use have made it a valuable tool in the fields of cell biology, immunology, and drug discovery.
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Total RNA was isolated from cells harvested after 24, 72 and 96 h using an RNeasy mini Kit (Qiagen Scientifics, MD) according to the manufacturer's instructions. Reverse transcription was carried out with an Iscript reverse transcription kit (BioRad, Hercules, CA). The resulting cDNA was used as a template for QRT-PCR, which was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and iCycler iQ Real-Time PCR Detection System (BioRad).
For c-MYB PCR the following primers were used: forward, dGAAGGTCGAACAGGAAGGTTATCT; and reverse, dGTAACGCTACAGGGTATGGAACA.
The c-MYB probe was labeled with reporter dye: 6-carboxyfluorescein (FAM) at the 5′ end and Black Hole quencher at 3′ end. The probe sequence was dTCAAAAGCCAGCCAGCCAGCAGTG.
The GAPDH was employed as a reporter gene for QRT-PCR. The product was obtained with the following primers: forward, dGACAGTCAGCCGCATCTTCTT; and reverse, dCCAATACGACCAAATCCGTTGAC.
The GAPDH probe was labeled with reporter dye: 6-carboxyfluorescein (FAM) at 5′ end and Black Hole quencher at 3′ end.
The probe sequence was dCGTCGCCAGCCGAGCCACATCG.
All reactions were performed in triplicates with 1 µl of cDNA. The volume of reaction mixture was 15 µl. The reaction mixture was pre-incubated at 50°C for 2 min. PCR cycling conditions were as follows: denaturation 95°C for 10 min, followed by 39 cycles 92°C for 15 s, 60°C for 45 s.
Analysis of QRT-PCR data was based on comparison of the target transcript PCR signal in a treatment group to signal measured in an untreated control. Analysis was done using the 2−ΔΔCT method as described by Levak and Schmittgen (31 (link)).
For c-MYB PCR the following primers were used: forward, dGAAGGTCGAACAGGAAGGTTATCT; and reverse, dGTAACGCTACAGGGTATGGAACA.
The c-MYB probe was labeled with reporter dye: 6-carboxyfluorescein (FAM) at the 5′ end and Black Hole quencher at 3′ end. The probe sequence was dTCAAAAGCCAGCCAGCCAGCAGTG.
The GAPDH was employed as a reporter gene for QRT-PCR. The product was obtained with the following primers: forward, dGACAGTCAGCCGCATCTTCTT; and reverse, dCCAATACGACCAAATCCGTTGAC.
The GAPDH probe was labeled with reporter dye: 6-carboxyfluorescein (FAM) at 5′ end and Black Hole quencher at 3′ end.
The probe sequence was dCGTCGCCAGCCGAGCCACATCG.
All reactions were performed in triplicates with 1 µl of cDNA. The volume of reaction mixture was 15 µl. The reaction mixture was pre-incubated at 50°C for 2 min. PCR cycling conditions were as follows: denaturation 95°C for 10 min, followed by 39 cycles 92°C for 15 s, 60°C for 45 s.
Analysis of QRT-PCR data was based on comparison of the target transcript PCR signal in a treatment group to signal measured in an untreated control. Analysis was done using the 2−ΔΔCT method as described by Levak and Schmittgen (31 (link)).
6-carboxyfluorescein
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Oligonucleotide Primers
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To optimize the RFADCC assay (Gomez-Roman et al., 2006 (link)) for high throughput efficiency, the regular double staining with the membrane PKH-26 dye and the intracellular carboxyfluorescein diacetate, succinimidyl ester (CSFE) dye were replaced with the membrane staining of the CCR5-SNAP-tag and the constitutive intracellular expression of GFP. For the ADCC protocol (Fig. 2 ), EGFP-CEM-NKr-CCR5-SNAP target cells were stained with the fluorescent substrate SNAP-Surface Alexa Fluor 647 (New England BioLabs Cat. S9136S) for 20 min at 37 °C with or without coating of monomeric HIV-1 Bal gp120 (50 μg/ml). For the studies with spinoculated virus, the cells were first stained with the SNAP-Surface dye and then spinoculated with the AT-2 inactivated Bal HIV-1 virus at 2000 RPM for 2 h at 12 °C. Gp120-sensitized, virus-spinoculated or infected EGFP-CEM-NKr-CCR5-SNAP target cells were then washed twice with cold R10 medium and added to a 96-well V-bottom plate (5000 cells/well). A final volume of 100 μl/well of antibody dilution was added and incubated with sensitized targets for 15 min at room temperature. A total of 250,000 purified human effector PBMC isolated by Ficoll-Paque from the whole blood of healthy human donors cells were added to each well at an effector/target (E/T) ratio of 50:1. After incubation at 37 °C in 5% CO2 for 2 h (gp120-coated) or 3 h (virus-spinoculated or infected cells), cells were washed with phosphate-buffered saline (PBS) containing 1% FBS (wash buffer), and fixed in 1% paraformaldehyde. Samples were analyzed at approximately 35,000 events per sample on an LSRII Fortessa flow cytometer (BD Biosciences). Doublets were excluded by forward-scatter area (FSC-A) versus forward-scatter height (FSC-H). Data were analyzed by FlowJo software (Tree Star, Ashland, OR). ADCC activity (=% cytotoxicity) is defined as the percentage of EGFP-CEM-NKr-CCR5-SNAP target cells that lose GFP staining but retain the CCR5-SNAP tag dye.
For cell surface staining, HIV-1 gp120-sensitized and infected EGFP-CEM-NKr-CCR5-SNAP target cells were incubated for 30 min at RT with 1 and 5 μg/ml, respectively, of Alexa-Fluor 647-conjugated mAbs C11, N5-i5 or N12-i2 in PBS. Target cells spinoculated with Bal AT-2 inactivated virus were incubated for 30 min at RT, respectively with 2 μg/ml of Alexa-Fluor 594-conjugated mAbs C11, N5-i5, N12-i2 or N10U1 in PBS. Cells were then washed once with wash buffer and fixed in a 1% paraformaldehyde.
For cell surface staining, HIV-1 gp120-sensitized and infected EGFP-CEM-NKr-CCR5-SNAP target cells were incubated for 30 min at RT with 1 and 5 μg/ml, respectively, of Alexa-Fluor 647-conjugated mAbs C11, N5-i5 or N12-i2 in PBS. Target cells spinoculated with Bal AT-2 inactivated virus were incubated for 30 min at RT, respectively with 2 μg/ml of Alexa-Fluor 594-conjugated mAbs C11, N5-i5, N12-i2 or N10U1 in PBS. Cells were then washed once with wash buffer and fixed in a 1% paraformaldehyde.
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Most recents protocols related to «Carboxyfluorescein»
The proliferation of CD19-CAR-iNKT cells and T cells was determined by carboxyfluorescein succinimidyl ester (CFSE) dilution. First, CD19-CAR-iNKT cells or MACS-isolated T cells were resuspended in phosphate-buffered saline (PBS, Gibco) and stained with CellTrace CFSE cell proliferation kit (BioLegend) for 5 min at room temperature. Immediately after staining, cells were washed with pure FBS and then two times in PBS supplemented with 5% FBS and finally resuspended in CAR medium. CFSE-labeled cells were tested in a mixed lymphocyte reaction (MLR) and against modified Raji cells. Where stated, 10 µg/mL nivolumab (Novartis) or its IgG4 isotype control (BioLegend) was added. Results are expressed as a percentage of proliferating cells or as mean fluorescence intensity.
Non-fluorescent liposomes were prepared as described above. 5(6)-Carboxyfluorescein (CF) (Merck; 100 mM CF stock, dissolved in 300 mM NaOH) was mixed in a 1:1 ratio with a buffer, containing 300 mM NaCl, and 100 mM Na2HPO4-NaH2PO4, pH 6. The dried lipid film was rehydrated with the 50 mM CF-stock solution, followed by sonication and extrusion. Finally, to remove the excess CF, the liposomes were applied onto a gravity flow desalting column (PD MiniTrap G25, Cytiva), pre-equilibrated with an elution buffer containing 150 mM NaCl, and 50 mM Na2HPO4-NaH2PO4, pH 6. Liposome content mixing fluorometry measurements were performed using a Varioskan LUX Fluorometer plate reader (ThermoFisher Scientific), similar to the R18-based assay in a 96-well format (200 µL reaction volume). Briefly, 490/515 nm excitation/emission wavelengths and 5 nm slits were used. The liposome mixture consisted of a 1:2 ratio between CF-encapsulating and empty LUVs (250 µM total lipid concentration), respectively, and measurements were performed in the elution buffer.
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Liposome membrane
integrity was studied by monitoring the fluorescence emitted from
CF upon release from liposomes. CF was encapsulated in liposomes at
self-quenching concentrations (50 mM) and fluorescence was observed
over time using a 96-well plate in a fluorescent plate reader (Tecan
Infinite M1000 Pro, Tecan Austria GmbH, Grödig/Salzburg, Austria)
at room temperature with λex = 485 nm and λem = 520 nm. Liposomes in PBS (10 mM, pH 7.4) were prepared,
and the fluorescence was measured (F0).
A peptide or enzyme was then added to the wells so that desired concentrations
were obtained. PBS (10 mM, pH 7.4) volumes equal to that was added
to the control wells. Control wells contained liposomes in PBS (10
mM, pH 7.4) and sample wells contained liposomes, peptide, and/or
PGA (Merck, Darmstadt, Germany) in PBS (10 mM, pH 7.4). All of the
wells had the same lipid concentration (40 μM). Fluorescence
measurements were performed every other minute over the desired time
span (F). After measurements, Triton X-100 was added
to the wells to achieve a 1% Triton concentration. After 10 min of
incubation to achieve complete liposome lysis, fluorescence was measured
(Ftot). Percentage of released CF was
calculated according to CF release (%) = (F − F0)/(Ftot − F0) × 100.
integrity was studied by monitoring the fluorescence emitted from
CF upon release from liposomes. CF was encapsulated in liposomes at
self-quenching concentrations (50 mM) and fluorescence was observed
over time using a 96-well plate in a fluorescent plate reader (Tecan
Infinite M1000 Pro, Tecan Austria GmbH, Grödig/Salzburg, Austria)
at room temperature with λex = 485 nm and λem = 520 nm. Liposomes in PBS (10 mM, pH 7.4) were prepared,
and the fluorescence was measured (F0).
A peptide or enzyme was then added to the wells so that desired concentrations
were obtained. PBS (10 mM, pH 7.4) volumes equal to that was added
to the control wells. Control wells contained liposomes in PBS (10
mM, pH 7.4) and sample wells contained liposomes, peptide, and/or
PGA (Merck, Darmstadt, Germany) in PBS (10 mM, pH 7.4). All of the
wells had the same lipid concentration (40 μM). Fluorescence
measurements were performed every other minute over the desired time
span (F). After measurements, Triton X-100 was added
to the wells to achieve a 1% Triton concentration. After 10 min of
incubation to achieve complete liposome lysis, fluorescence was measured
(Ftot). Percentage of released CF was
calculated according to CF release (%) = (F − F0)/(Ftot − F0) × 100.
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LUV1000 of a final concentration of 10 μM in 2 mL volume were treated with a range of LEGO-LPPO concentrations (0.1–12.5 mg L−1). The increase of fluorescence intensity (F, excitation 480 nm, emission 515 nm, band paths 2 nm) induced by leakage of CF content from the liposomes was recorded over time and normalized to the maximum intensity (Fmax) induced by lysing the vesicles with 0.1% Triton X-100. CF leakage was quantified using the following equation: % CF leakage = (F − F0)/(Fmax − F0) × 100% where F is an actual fluorescence intensity, F0 is the fluorescence intensity before the addition of permeabilizing agent and Fmax is the maximum fluorescence caused by complete disruption of all liposomes by Triton X-100.
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The 96-well plates were precoated with 10 µg/mL anti-mouse CD3 mAb (BioLegend) and 5 µg/mL anti-mouse CD28 mAb (BioLegend) to stimulate CD4+T-cells. The total CD4+T-cells were isolated from spleens of naive mice by positive CD4+T-cell isolation kit (Miltenyi Biotec, USA). The cells were then labeled with 1 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) for 10 min and co-cultured with MSC-sEVs at a concentration of 10 µg/ml. After 72 h of incubation, the CFSE fluorescence intensity was measured by FACS and analyzed by FlowJo.
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Top products related to «Carboxyfluorescein»
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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.
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CFSE (Carboxyfluorescein Succinimidyl Ester) is a fluorescent dye used in cell biology for tracking cell division and proliferation. It covalently binds to intracellular proteins, allowing the dye to be equally distributed between daughter cells during cell division.
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CFSE is a fluorescent dye used for cell labeling and tracking. It binds to cellular proteins, allowing researchers to monitor cell division and proliferation over time.
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The CellTrace CFSE Cell Proliferation Kit is a lab equipment product for tracking cell division and proliferation. It uses a fluorescent dye to label cells, enabling the monitoring of cell division over time.
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The FACSCalibur is a flow cytometry system designed for multi-parameter analysis of cells and other particles. It features a blue (488 nm) and a red (635 nm) laser for excitation of fluorescent dyes. The instrument is capable of detecting forward scatter, side scatter, and up to four fluorescent parameters simultaneously.
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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CFSE (Carboxyfluorescein Succinimidyl Ester) is a fluorescent dye used for cell labeling and tracking. It binds to cellular proteins, allowing the visualization and quantification of cell division and proliferation.
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RPMI 1640 medium is a commonly used cell culture medium developed at Roswell Park Memorial Institute. It is a balanced salt solution that provides essential nutrients, vitamins, and amino acids to support the growth and maintenance of a variety of cell types in vitro.
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The FACSCalibur flow cytometer is a compact and versatile instrument designed for multiparameter analysis of cells and particles. It employs laser-based technology to rapidly measure and analyze the physical and fluorescent characteristics of cells or other particles as they flow in a fluid stream. The FACSCalibur can detect and quantify a wide range of cellular properties, making it a valuable tool for various applications in biology, immunology, and clinical research.
More about "Carboxyfluorescein"
Carboxyfluorescein (CF, CFSE) is a widely-used fluorescent dye and tracer in biological and medical research.
It is a derivative of the fluorescein molecule, with a carboxyl group attached.
This versatile dye is commonly employed to label cells, proteins, and other biomolecules, allowing their visualization and tracking in various experimental settings.
Carboxyfluorescein's excitation and emission wavelengths make it compatible with common fluorescence detection equipment, such as the FACSCalibur and FACSCanto II flow cytometers.
It is often used in assays to measure important cellular processes, including viability, proliferation, and more.
The CellTrace CFSE Cell Proliferation Kit leverages the properties of carboxyfluorescein to assess cell division and track cell populations over time.
Researchers in fields like cell biology, immunology, and drug discovery have found carboxyfluorescein to be a valuable tool due to its versatility and ease of use.
It can be used to label cells cultured in RPMI 1640 medium supplemented with FBS, allowing for effective tracking and analysis.
To optimize your carboxyfluorescein-based assays, consider using PubCompare.ai, an AI-driven research protocol comparison tool.
This innovative solution can help you locate the best protocols from literature, preprints, and patents, and compare them side-by-side to improve reproducibility and identify the most reliable and effective protocols for your specific research needs.
It is a derivative of the fluorescein molecule, with a carboxyl group attached.
This versatile dye is commonly employed to label cells, proteins, and other biomolecules, allowing their visualization and tracking in various experimental settings.
Carboxyfluorescein's excitation and emission wavelengths make it compatible with common fluorescence detection equipment, such as the FACSCalibur and FACSCanto II flow cytometers.
It is often used in assays to measure important cellular processes, including viability, proliferation, and more.
The CellTrace CFSE Cell Proliferation Kit leverages the properties of carboxyfluorescein to assess cell division and track cell populations over time.
Researchers in fields like cell biology, immunology, and drug discovery have found carboxyfluorescein to be a valuable tool due to its versatility and ease of use.
It can be used to label cells cultured in RPMI 1640 medium supplemented with FBS, allowing for effective tracking and analysis.
To optimize your carboxyfluorescein-based assays, consider using PubCompare.ai, an AI-driven research protocol comparison tool.
This innovative solution can help you locate the best protocols from literature, preprints, and patents, and compare them side-by-side to improve reproducibility and identify the most reliable and effective protocols for your specific research needs.