Cfda se

Manufactured by BD

Sourced in United States

CFDA-SE is a fluorescent cell labeling reagent used for cell tracking and proliferation assays in flow cytometry applications. It covalently binds to cellular proteins, allowing for the monitoring of cell division over time.

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Lab products found in correlation

Cfda se, by Thermo Fisher Scientific (4 mentions) Human il 2, by R&D Systems (2 mentions) Lsr 2 cytometer, by BD (2 mentions) Anti cd3 cd28 beads, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by GE Healthcare (2 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions) Anti cd3 ucth 1 mab, by BD (1 mentions) 7 aminoactinomycin d 7 aad, by BD (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Facscalibur, by BD (1 mentions) Cellquest pro software, by BD (1 mentions) V plex assay, by Mesoscale (1 mentions) Anti cd107a bv421, by BioLegend (1 mentions) Anti cd3, by BioLegend (1 mentions) Cytotoxicity detection kitplus, by Merck Group (1 mentions) Anti cd8, by BD (1 mentions) Anti cd4, by BD (1 mentions)

8 protocols using cfda se

Evaluating CD8+ T Cell Suppression

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The suppressive activity of Treg was evaluated by monitoring the inhibition of dye dilution in PBMC stained with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, 5 µM, Molecular Probes, Invitrogen).
Briefly, the PBMC-CFDA-SE+ were pulsed with the anti-CD3 UCTH-1 mAb (5 µg/ml, BD Bioscience) and cultured for 5 days in a 96-well U bottomed plate (1 × 10⁵ cells/well) in the presence (or not) of ex vivo- or in vitro-generated CD8+ T lymphocytes (1 × 10⁵ cells/well). Then, the samples were harvested, washed in PBS, and analyzed by flow cytometry. The dead cells were excluded from analysis by adding 7-aminoactinomycin D (7-AAD) (BD Bioscience) prior to analysis. Suppression activity was expressed as the percentage reduction of the proliferation in the presence of CD8+ Treg lymphocytes compared to the levels of proliferation observed in control cultures of PBMCs cultured in the absence of Treg cells. A suppression activity ≥25% was considered significant. This threshold was chosen based on the results achieved in a large historical cohort of more than 50 healthy subjects of both sexes with age ranging from 18 to 87 years. In healthy donors, CD8+ Treg suppression activity never fell below 25%.

Negrini S., Fenoglio D., Parodi A., Kalli F., Battaglia F., Nasi G., Curto M., Tardito S., Ferrera F, & Filaci G. (2017). Phenotypic Alterations Involved in CD8+ Treg Impairment in Systemic Sclerosis. Frontiers in Immunology, 8, 18.

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Measuring MDSC-Mediated T Cell Suppression

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FACS was used to isolate T cells, PMN-MDSCs, M-MDSCs, and classical monocytes based on the following respective membrane phenotypes: CD3⁺CD19^-; HLA-DR^loCD11b⁺CD33⁺CD15⁺; HLA-DR^loCD11b⁺CD33⁺CD14⁺ and (CD11b⁺CD14⁺HLA-DR⁺). To measure MDSC-induced suppression of autologous T-lymphocyte proliferation, after exposing T cells to carboxyfluorescein diacetate succinimidyl ester (CFDA-SE 1 μM; ThermoFisher) for 10 minutes at 37°C, they were cultured with or without MDSCs in a 2:1 ratio, respectively. T-cell proliferation was measured by the extent of CFDA-SE dilution using a BD LSRII cytometer. Cultures were carried out in complete medium consisting of RPMI1640 (Gibco), 10% FBS (Atlanta Biologicals), 1x Penicillin-Streptomycin (GE Healthcare Life Sciences), GlutaMAX (Gibco) supplemented with human IL-2 (50u/ml; R&D) and anti-CD3/CD28 beads (Thermo Scientific) according to the manufacture’s recommendation for 72 hours.

Ferrer G., Jung B., Chiu P.Y., Aslam R., Palacios F., Mazzarello A.N., Vergani S., Bagnara D., Chen S.S., Yancopoulos S., Xochelli A., Yan X.J., Burger J.A., Barrientos J.C., Kolitz J.E., Allen S.L., Stamatopoulos K., Rai K.R., Sherry B, & Chiorazzi N. (2021). Myeloid-derived suppressor cell subtypes differentially influence T-cell function, T-helper subset differentiation, and clinical course in CLL. Leukemia, 35(11), 3163-3175.

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Flow Cytometry Isolation and Functional Assay of Immune Cells

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FACS was used to isolate T cells, PMN-MDSCs, M-MDSCs, and classical monocytes based on the following respective membrane phenotypes: CD3⁺CD19⁻; HLA-DR^loCD11b⁺CD33⁺CD15⁺; HLA-DR^loCD11b⁺CD33⁺CD14⁺ and (CD11b⁺CD14⁺HLA-DR⁺). To measure MDSC-induced suppression of autologous T-lymphocyte proliferation, after exposing T cells to carboxyfluorescein diacetate succinimidyl ester (CFDA-SE 1 µM; ThermoFisher) for 10 min at 37 °C, they were cultured with or without MDSCs in a 2:1 ratio, respectively. T-cell proliferation was measured by the extent of CFDA-SE dilution using a BD LSRII cytometer. Cultures were carried out in complete medium consisting of RPMI1640 (Gibco), 10% FBS (Atlanta Biologicals), 1× penicillin–streptomycin (GE Healthcare Life Sciences), GlutaMAX (Gibco) supplemented with human IL-2 (50 µ/ml; R&D), and anti-CD3/CD28 beads (Thermo Scientific) according to the manufacture’s recommendation for 72 h.

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Live/Dead Assay with CFDA-SE and Propidium Iodide

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Cells were harvested from culture and washed in sterile warm PBS-G. The pellet was resuspended in PBS-G with 10 μM CFDA-SE (ThermoFisher), and incubated for 15 mins at 37°C. Cells were washed with HMI-9 medium and incubated in HMI-9 for 30 mins at 37°C. Cells were then washed, and fixed with 3.7% (v/v) formaldehyde for 10 min (a detailed paper on validating CFDA-SE staining as a live/dead assay compatible with fixation of cells will be published elsewhere). Fixative was washed out with PBS-G, and cells were resuspended in PBS-G plus 5 μg/ml Hoechst 33342 (Life Tech.). Samples were analysed with excitation peak of 492 nm and emission peak of 517 nm for CFDA-SE on a BD LSRII instrument.
For propidium iodide (PI) staining, 1 μl 500 ng/ml PI was added to the final resuspension before analysis. Samples were analysed at peak excitation at 488 nm and emission at 695 nm.

Dewar C.E., MacGregor P., Cooper S., Gould M.K., Matthews K.R., Savill N.J, & Schnaufer A. (2018). Mitochondrial DNA is critical for longevity and metabolism of transmission stage Trypanosoma brucei. PLoS Pathogens, 14(7), e1007195.

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CFDA-SE Proliferation Assay for Tumor-Bearing Mice

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For carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) proliferation assay, lymphocytes from spleens of MBT-2-luc tumor-bearing mice with different treatments were incubated for 15 min in the darkness with 5 μM CFDA-SE (Thermo Fisher Scientific, Waltham, MA, USA) in PBS and then washed. The assay was performed by co-culturing 1 × 10⁵ target xenogeneic urothelial cells or MBT-2-luc cells together with 5 × 10⁵ CFDA-SE-labeled lymphocytes from spleens (E/T ratio 5:1) for 2 days. The intensity of CFDA-SE fluorescence in lymphocytes was measured by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo Software.

Huang C.P., Wu C.C, & Shyr C.R. (2020). Combination of novel intravesical xenogeneic urothelial cell immunotherapy and chemotherapy enhances anti-tumor efficacy in preclinical murine bladder tumor models. Cancer Immunology, Immunotherapy, 70(5), 1419-1433.

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Evaluation of Cell Cycle and Proliferation

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For the analysis of DNA ploidy and cell cycle, cells were grown, harvested and counted. One million cells were fixed in 70% ethanol, followed by staining with propidium iodide (PI) diluted in PBS pH 7.4 (PI 200 μg/ml, RNAse A 20 mg/ml, 0.1% Triton X-100) for 15 min at 37°C. For proliferation assays harvested cells were counted and 1x10⁶ were stained with 5μM Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Sigma-Aldrich), for 30min at 37°C with shaking. The reaction was stopped with two sequential wash steps with complete culture media and cells returned to culture afterwards. All samples were acquired in a FACSCalibur (Becton Dickinson, Burlington, MA) flow cytometer equipped with CellQuest Pro Software, (Becton Dickinson, San Jose, CA) using the FL1 (CFDA-SE) and FL2 (PI) channels. Analyses of DNA ploidy, cell cycle and proliferation were performed using the FlowJo software version 7.6.4 (Tree Star Inc, San Carlos, CA).

Gentile L.B., Nagamine M.K., Biondi L.R., Sanches D.S., Toyota F., Giovani T.M., de Jesus I.P., da Fonseca I.I., Queiroz-Hazarbassanov N., Diaz B.L., Salles Gomes C.D, & Dagli M.L. (2017). Establishment of primary mixed cell cultures from spontaneous canine mammary tumors: Characterization of classic and new cancer-associated molecules. PLoS ONE, 12(9), e0184228.

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Multiparametric Analysis of T-Cell Functionality

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Cytotoxicity, activation, degranulation, and proliferation of T cells, and cytokine secretion were analyzed in multiparametric cytotoxicity assays. Cell lysis was assessed by quantification of lactate dehydrogenase concentrations in the cell culture supernatant using the Cytotoxicity Detection Kit^PLUS (Sigma-Aldrich) according to manufacturer's instructions. Activation and degranulation of T cells were assessed by staining cells for extracellular CD25 (anti-CD25-APC, Becton Dickinson, RRID:AB_2916552), CD69 (anti-CD69-PE-Cy7, Becton Dickinson, RRID:AB_396851), and CD107a (anti-CD107a-BV421, BioLegend, RRID:AB_11203537), and intracellular granzyme B (anti-GrzB-FITC, Becton Dickinson, RRID:AB_1645488) by flow cytometry. For analysis of T-cell proliferation, the PBMCs were labeled with CFDA-SE (Becton Dickinson) and T cells were additionally stained with anti-CD3 (BioLegend, RRID:AB_2561628). Cytokine concentrations were analyzed from supernatants of cytotoxicity assays using the V-PLEX Assays (Mesoscale Discovery) following the manufacturer's instructions.

Zhang W., Auguste A., Liao X., Walterskirchen C., Bauer K., Lin Y.H., Yang L., Sayedian F., Fabits M., Bergmann M., Binder C., Corrales L., Vogt A.B., Hudson L.J., Barnes M.P., Bisht A., Giragossian C., Voynov V., Adam P.J, & Hipp S. (2022). A Novel B7-H6–Targeted IgG-Like T Cell–Engaging Antibody for the Treatment of Gastrointestinal Tumors. Clinical Cancer Research, 28(23), 5190-5201.

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Cytotoxicity Assay of T-cells

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Redirected T-cell cytotoxicity was assayed by flow cytometry using human PBMCs (peripheral blood mononuclear cells) or purified T cells and various tumor cell lines or primary myeloma cells. Effector cells were co-incubated with target cells and serial dilutions of BI 836909. After incubation, antihuman CD138 antibody or anti-CD33 (for HL-60 cells) antibody (Miltenyi Biotec, San Diego, CA, USA) was added to the cells to distinguish target from effector cells. For detection of T-cell activation, the cells were stained with anti-CD4, anti-CD8, anti-CD25 and anti-CD69 antibodies (all from Becton Dickinson, Franklin Lakes, NJ, USA). For analysis of T-cell proliferation, the T cells were labeled with CFDA-SE (Becton Dickinson). The cells were analyzed by flow cytometry. Number of depleted target cells has been determined with the following formula: % Lysis = 100 -(viable cells of treatment group × 100/viable cells of untreated control group).

Hipp S., Tai Y.T., Blanset D., Deegen P., Wahl J., Thomas O., Rattel B., Adam P.J., Anderson K.C, & Friedrich M. (2017). A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo. Leukemia, 31(8).

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