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Celltracetm cfse

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CellTraceTM CFSE is a cell-permeable fluorescent dye that binds to proteins within cells. It can be used to monitor cell division and track cell proliferation in vitro.

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Lab products found in correlation

Celltracetm far red, by Thermo Fisher Scientific (2 mentions) Celltracetm violet, by Thermo Fisher Scientific (2 mentions) Cell proliferation kit, by Thermo Fisher Scientific (2 mentions) Anti cd3 mab clone 145 2c11, by BD (1 mentions) Imagexpress micro microscope, by Molecular Devices (1 mentions) Human cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Vybrant dyecycletm, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Anti cd3e, by Thermo Fisher Scientific (1 mentions) Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions) Efluor 780, by Thermo Fisher Scientific (1 mentions) Transwell, by BD (1 mentions) Matrigel matrix, by BD (1 mentions) Cytoflex s flow cytometer, by Beckman Coulter (1 mentions) 7 aad, by Beckman Coulter (1 mentions) Macsquant analyzer, by Miltenyi Biotec (1 mentions)

20 protocols using celltracetm cfse

1

Fluorescent Dye Staining Protocol

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For staining, CellTraceTM Violet, CellTraceTM CFSE and CellTraceTM Far Red fluorescent dyes (Thermo Fisher Scientific, Waltham, MA, United States) were prepared according to the manufacturers’ recommendations. Cultures were stained with 2 μL CellTraceTM dye per mL culture and incubated overnight in the dark at 12°C and 200 RPM. Stained cultures were washed twice with YP medium, as remaining unbound dye molecules would bind to the amide groups in yeast extract and peptone.
Gorter de Vries A.R., Koster C.C., Weening S.M., Luttik M.A., Kuijpers N.G., Geertman J.M., Pronk J.T, & Daran J.M. (2019). Phenotype-Independent Isolation of Interspecies Saccharomyces Hybrids by Dual-Dye Fluorescent Staining and Fluorescence-Activated Cell Sorting. Frontiers in Microbiology, 10, 871.
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2

CFSE-Based B Cell Proliferation Assay

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Freshly isolated B cells were labeled with 0.5 μM CellTraceTM CFSE (ThermoFisher, Waltham, MA, USA) and incubated for 10 min at 37 °C in the dark. Washed CFSE-labeled cells were stimulated and cultured at 37 °C/5% CO2. Four or six days later, B cell proliferation was evidenced by a cell division-dependent decrease in CFSE staining intensity as evaluated by flow cytometry (Fig. S1). Fluorescence data were analyzed with CXP (Beckman Coulter, Fullerton, CA) and FlowJo v.8.7 (TreeStar, Ashland, USA) softwares.
Schleiss C., Ilias W., Tahar O., Güler Y., Miguet L., Mayeur-Rousse C., Mauvieux L., Fornecker L.M., Toussaint E., Herbrecht R., Bertrand F., Maumy-Bertrand M., Martin T., Fournel S., Georgel P., Bahram S, & Vallat L. (2019). BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo. Scientific Reports, 9, 701.
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3

T Cell Proliferation Assay with CFSE Labeling

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WT and lamp2 KO-derived CD4 and CD8 T cells were sorted, labeled with 1 μM of CellTraceTM CFSE (carboxyfluorescein diacetate succinimidyl ester; ThermoFisher Scientific, C34554) and cultured in the presence of 1 μg/ml anti-CD3 mAb (clone 145–2C11; BD Pharmingen, 553,057) and 5 μg/ml anti-C28 mAb (clone 37.51; BD Pharmingen, 553,294). The precursor frequency of dividing cells (percentage of cells in the initial population that undergone one or more divisions after culture) was calculated as follows: [∑n ≥ 1(Pn/2 n)]/[∑n ≥ 0(Pn/2 n)], where n is the division number that cells have gone through and Pn is the number of cells in division, as described [56 (link)].
Rodrigues P.M., Sousa L.G., Perrod C., Maceiras A.R., Ferreirinha P., Pombinho R., Romera-Cárdenas G., Gomez-Lazaro M., Senkara M., Pistolic J., Cabanes D., Klein L., Saftig P, & Alves N.L. (2022). LAMP2 regulates autophagy in the thymic epithelium and thymic stroma-dependent CD4 T cell development. Autophagy, 19(2), 426-439.
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4

Evaluating Tumor Cell Migration through Endothelial Barrier

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To evaluate MDA-MB-231 cell migration through a layer of endothelial cells, 8 × 104 HUVECs were subcultured onto 8.0 μm pore 12-well inserts (Greiner Bio-one®, Frickenhausen, Germany Catalog number: C34554) with serum in the upper and lower chambers for 24 h in 5% CO2 at 37 °C. Then, MDA-MB-231 cells (0.6 × 105) were labeled with Cell Trace TM CFSE (Thermo Fisher Scientific, Waltham, MA, USA, Catalog number: C34554). MDA-MB-231 cells were treated with DisBa-01 in serum-free medium for 30 min at room temperature, and then allowed to transmigrate through the endothelial layer for 16 h at 37 °C in a normoxic and hypoxic environment. The lower chamber contained medium plus 10% SFB. Filters were fixed with 3.7% paraformaldehyde and the remaining cells on the upper surface were removed using a cotton swab. The nuclei of migrated cells were stained with 0.7 ng/µL DAPI solution (Thermo Fisher Scientific, Waltham, MA, USA, Catalog Number: 62248). Membranes were assembled on a microscope slide for automated cell counting in an ImageXpress Micro microscope (Molecular Devices San Jose, CA, USA) under 10× magnification with the Meta-X-press software, quantified using the Multi Wavelength Cell Scoring.
Casali B.C., Gozzer L.T., Baptista M.P., Altei W.F, & Selistre-de-Araújo H.S. (2022). The Effects of αvβ3 Integrin Blockage in Breast Tumor and Endothelial Cells under Hypoxia In Vitro. International Journal of Molecular Sciences, 23(3), 1745.
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5

Homing of FTVI-treated Monocyte-derived Dendritic Cells

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Human mo-DCs were harvested and either FTVI- or buffer-treated as described above. Mo-DCs were then suspended in RPMI medium at 1×107 cells/mL and split into two groups for dye labeling, with either 10 µM CellTraceTM CFSE or CellTraceTM Far Red (Thermo Fisher Scientific, Grand Island, NY, USA), for 20 min at 37°C. Subsequently, cells were washed 3 times with ice-cold RPMI supplemented with 10% fetal bovine serum. Reciprocal mixtures of 1:1 (FTVI-treated DCs and buffer treated-DCs) were prepared for each dye combination. Recipient NSG mice were intravenously co-injected (retro-orbitally) with 2.5×106 FTVI-treated mo-DCs or 2.5×106 buffer-treated mo-DCs in a volume of 0.1 mL of PBS. After 24 h, both tibias and femurs were removed, and the bone marrow was flushed. The collected cell suspensions were strained, washed and erythrocytes were lysed in NH4Cl buffer. The presence of homed mo-DCs in bone marrow was assessed by flow cytometry of dye-labeled cells. The data collected were analyzed with FlowJo software version 10.0.5 (TreeStar, San Carlos, CA, USA). Fold change of FTVI-treated mo-DCs compared to buffer-treated mo-DCs was calculated for each mouse (6 mice, 3 mice for each dye combination).
Videira P.A., Silva M., Martin K.C, & Sackstein R. (2018). Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-derived Dendritic Cells Programs Transendothelial Migration. Journal of immunology (Baltimore, Md. : 1950), 201(3), 1030-1043.
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6

Salivary Exosome Effect on CD4+ T Cell Proliferation

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The method was described before in previous publications (10 (link)). Briefly, CD4+ T cells were isolated from PBMCs of healthy donors by use of the human CD4+ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-096-533) according to the manufacturer's instruction. For CFSE staining, CD4+ T cells were incubated with 5 μM CellTraceTM CFSE (Thermo Fisher Scientific, Waltham, MA, USA, C34554) at 37°C for 20 min in the dark, according to manufacturer's instructions. CD4+ T cells were activated as described above and co-incubated after 24 h with 30 μg salivary exosomes for 4 d. As a positive control, plasma-derived exosomes from HNSCC patients were used (10 (link)). Proliferation of CD4+ T cells was measured by flow cytometry.
Hofmann L., Medyany V., Ezić J., Lotfi R., Niesler B., Röth R., Engelhardt D., Laban S., Schuler P.J., Hoffmann T.K., Brunner C., Jackson E.K, & Theodoraki M.N. (2022). Cargo and Functional Profile of Saliva-Derived Exosomes Reveal Biomarkers Specific for Head and Neck Cancer. Frontiers in Medicine, 9, 904295.
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7

Tracking Cell Proliferation and Morphology with CFSE

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Carboxyfluorescein diacetate succinimidyl ester (CellTrace TM CFSE, ThermoFisher) was used to label cells to track proliferation and assess morphology. For proliferation assays from fresh samples and to assess the distribution of cells within the electrospun scaffolds, 3T3 or C2C12 cells were pre-stained with CFSE stock as per the manufacturer's protocol and then electrospun as described previously. For freeze-thaw proliferation assays, pre-encapsulated 3T3 cells were stained one day following the thawing of the scaffold, again using the same protocol but adding 4
x 3 mL washes in PBS to ensure any stain not taken up by cells is removed from the hydrogel scaffold. For cell morphology assays, pre-loaded 3T3 or C2C12 were stained with CFSE (using the recommended protocol with the additional washes) on the day the morphology assessment was conducted (i.e. following either 1, 5, 7 and 18 days of cell culture). In this latter assay, paraformaldehyde stock (PFA, Sigma-Aldrich, 3 wt%) was used to fix the cells prior to imaging.
Xu F., Dodd M., Sheardown H, & Hoare T. (2018). Single-Step Reactive Electrospinning of Cell-Loaded Nanofibrous Scaffolds as Ready-to-Use Tissue Patches. Biomacromolecules, 19(11).
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8

Multimodal Proliferation Tracking

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In addition to CFSE, Far red (CellTrace TM CFSE, ThermoFisher) was used to label cells to track proliferation within the freeze-thawed cell-loaded scaffolds. Cells were stained with Far Red stock solution following the manufacturer's protocol at the first day after thawing, after which the same imaging procedure as outlined for CFSE tracking was conducted.
Xu F., Dodd M., Sheardown H, & Hoare T. (2018). Single-Step Reactive Electrospinning of Cell-Loaded Nanofibrous Scaffolds as Ready-to-Use Tissue Patches. Biomacromolecules, 19(11).
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9

Cytolytic Assay of CAR T Cells

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The cytolytic activity of engineered T-cells endowed with the different CARs (transfected with dose D1/4), was assessed using a flow cytometry-based cytotoxicity assay. In this assay target cells presenting the CAR target antigen (Daudi, CD19pos) are labelled with CellTraceTM CFSE (Life Technologies) and control cells not presenting the CAR target antigen (K562, CD19neg) with CellTraceTM violet (Life Technologies). The target cell populations were co-incubate at 37 °C at various ratio of engineered effector CAR T cells (Effector/Target ratio of 10:1, 20:1 and 30:1) in a final volume of X-Vivo-15 media of 100 uL, for 5 h in presence of vehicle (Ethanol) or AP21967 (100 nM).
The whole cell population was recovered, washed in PBS and labeled with eFluor780 viability marker before being fixed by 2% PFA. Fixed cells were analyzed by flow cytometry to determine their viability.
Juillerat A., Marechal A., Filhol J.M., Valton J., Duclert A., Poirot L, & Duchateau P. (2016). Design of chimeric antigen receptors with integrated controllable transient functions. Scientific Reports, 6, 18950.
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10

Visualizing T-cell and EC Dynamics

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To visualize dynamics of T cells and nuclei of ECs, a confluent monolayer of bEnd.3 cells treated with TNF-α and SDF-1α were stained with Hoechst 33342 (5 μg/ml, Life Technologies) for 20 min. Then, DO11.10 T cell blasts labeled with CellTraceTM CFSE (1 μM, Life Technologies) or Vybrant DyeCycleTM (0.5 μM, Life Technologies) were perfused on Hoechst-labeled bEnd.3 cells and each fluorescence image was sequentially acquired by time-lapse imaging for 15 min with 20 s interval.
Song K.H., Lee J., Park H., Kim H.M., Park J., Kwon K.W, & Doh J. (2016). Roles of endothelial A-type lamins in migration of T cells on and under endothelial layers. Scientific Reports, 6, 23412.
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