Cell proliferation dye efluor 670

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The Cell Proliferation Dye eFluor 670 is a fluorescent dye used for tracking cell division and proliferation in vitro. The dye binds to cellular proteins, and its fluorescence intensity is diluted with each cell division, allowing for the monitoring of cell proliferation through flow cytometry analysis.

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98 protocols using cell proliferation dye efluor 670

Embryonic Cell Fate Modulation

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Distinct subpopulations of E7.5 or E8.5 embryonic Actb^EGFP+ cells were sorted based on their TLR2, c-kit, and CD45 surface expression, plated on a semi-confluent layer of OP-9 cells^{54 (link)} (gift from J.C. Zuniga-Pflucker) or ST-2 cells^{55 (link)} (gift from L. Klein) and cultured in RPMI containing 5% FCS (Sigma-Aldrich). E7.5 sorted cells were supplemented with recombinant cytokines IL-3 (1 ng/ml), SCF (50 ng/ml) G M-CSF (3 ng/ml), and M-CSF (10 ng/ml) (Biolegend). A thousand cells from each E8.5 sorted Actb^EGFP+ population labeled with Cell Proliferation Dye eFluor^® 670 (eBioscience) were co-cultured with OP-9 cells in the presence or absence of 1 μg/ml Pam₃CSK₄ (Invivogen) for 72 h. The total number of surviving Actb^EGFP+ cells were normalized to 10,000 events obtained by FCM analysis. E8.5 Tlr2^CreEYFP⁺ Lin^– cells were sorted, stained with Cell Proliferation Dye eFluor^® 670 (eBioscience) and cultured in the presence or absence of Pam₃CSK₄ (1 µg/ml) on ST-2 stroma. The proliferation history of embryonic cells was determined by the dilution of Cell Proliferation Dye^® eFluor 670 according to the manufacturer’s instructions.

Balounová J., Šplíchalová I., Dobešová M., Kolář M., Fišer K., Procházka J., Sedlacek R., Jurisicova A., Sung H.K., Kořínek V., Alberich-Jorda M., Godin I, & Filipp D. (2019). Toll-like receptor 2 expression on c-kit+ cells tracks the emergence of embryonic definitive hematopoietic progenitors. Nature Communications, 10, 5176.

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Thymocyte Labeling and Imaging

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Thymuses were collected from OT-I Rag2^-/-, F5 Rag1^-/-, or B6 mice and dissociated through a 70 μm cell strainer to yield a cell suspension. Thymocytes were then labeled with 1 μM Cell Proliferation Dye eFluor450 or 0.5 μM Cell Proliferation Dye eFluor670 (Thermo Fisher Scientific) at 10⁷ cells/ml at 37°C for 15 min in PBS, then washed and resuspended in complete RPMI (containing 10% FBS, penicillin streptomycin, and 2-mercaptoethanol, cRPMI) for overlay onto thymic slices. Thymocytes do not proliferate at the timepoints collected, allowing overlaid thymocytes to be distinguished from slice resident thymocytes by Cell Proliferation Dyes (Figure 1a). In imaging experiments, OT-I thymocytes were depleted of mature CD8 single positives using the EasySep Biotin Positive Selection Kit (Stemcell Technologies) with anti-human/mouse β7 integrin antibody (FIB504, Biolegend) according to the manufacturer’s instructions. Thymocytes were then labeled with 3 μM SNARF (Thermo Fisher Scientific) at 10⁷ cells/ml at 37°C for 15 min in PBS, then washed and labeled with 5 μM Hoechst 33342 (Thermo Fisher Scientific) at 10⁷ cells/ml at 37°C for 15 min. For all other experiments, the total, unfractionated thymocyte population was used.

Kurd N.S., Lutes L.K., Yoon J., Chan S.W., Dzhagalov I.L., Hoover A.R, & Robey E.A. (2019). A role for phagocytosis in inducing cell death during thymocyte negative selection. eLife, 8, e48097.

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Evaluating CAR-T Cell Cytotoxicity Against VEGFR2+ Targets

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EL4 cells that do not express VEGFR2 were labeled with Tag-It Violet Proliferation Cell Tracking Dye (Biolegend), and VEGFR2⁺ EL4 cells were labeled with Cell Proliferation Dye eFluor 670 (Thermo Fisher Scientific). Mouse CAR-T cells 4 days after Rv transduction, EL4 cells, and VEGFR2⁺ EL4 cells were co-cultured at the indicated E/T ratios. After 18 h, CountBright Absolute Cell Counting Beads (Thermo Fisher Scientific) and 7-AAD Viability Staining Solution (Biolegend) were added to the reaction wells, and the number of each target cell was analyzed using flow cytometry until 1000 beads were detected. Cytotoxicity was calculated using the following formula: Percentage of antigen-specific lysis = [(VEGFR2⁺ EL4 cells/ EL4 cells ratio in non-effector cell’s well) − (VEGFR2⁺ EL4 cells/ EL4 cells ratio in effector cell’s well)] / [VEGFR2⁺ EL4 cells / VEGFR2⁻ EL4 cells ratio in non-effector cell’s well] × 100.

Fujiwara K., Kitaura M., Tsunei A., Kusabuka H., Ogaki E, & Okada N. (2021). Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals. International Journal of Molecular Sciences, 22(5), 2476.

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FACS-based ADCC Assay for HIV-1

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Measurement of ADCC using a fluorescence-activated cell sorting (FACS)-based infected cell elimination (ICE) assay was performed at 48 h post-infection. Briefly, HIV-1-infected primary CD4+ T cells were stained with AquaVivid viability dye and cell proliferation dye eFluor670 (Thermo Fisher Scientific) and used as target cells. Cryopreserved autologous PBMC effectors cells, stained with cell proliferation dye eFluor450 (Thermo Fisher Scientific), were added at an effector: target ratio of 10:1 in 96-well V-bottom plates (Corning, Corning, NY). A 1:1000 final dilution of plasma or 5 μg/mL of anti-Env mAbs was added to appropriate wells and cells were incubated for 5 min at room temperature. The plates were subsequently centrifuged for 1 min at 300 × g, and incubated at 37°C, 5% CO₂ for 5 h before being fixed in a 2% PBS-formaldehyde solution. Infected cells were identified by intracellular p24 staining as described above. Samples were acquired on an LSR II cytometer (BD Biosciences) and data analysis was performed using FlowJo v10.5.3 (Tree Star). The percentage of ADCC was calculated with the following formula: [(% of p24 + cells in Targets plus Effectors) − (% of p24 + cells in Targets plus Effectors plus plasma)/(% of p24 + cells in Targets) × 100] by gating on infected lived target cells.

Prévost J., Anand S.P., Rajashekar J.K., Zhu L., Richard J., Goyette G., Medjahed H., Gendron-Lepage G., Chen H.C., Chen Y., Horwitz J.A., Grunst M.W., Zolla-Pazner S., Haynes B.F., Burton D.R., Flavell R.A., Kirchhoff F., Hahn B.H., Smith AB I.I.I., Pazgier M., Nussenzweig M.C., Kumar P, & Finzi A. (2022). HIV-1 Vpu restricts Fc-mediated effector functions in vivo. Cell reports, 41(6), 111624.

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Investigating CD8+ T cell regulation by MDSCs

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CD8⁺ T cells were recovered from the spleens using magnetic sorting (Cat.480035, BioLegend, USA) and activated by the addition of CD3 (Cat.100238, BioLegend, USA, 10 ng/mL) and CD28 (Cat.102116, BioLegend, USA, 5 ng/mL). After labeling with the Cell Proliferation Dye eFluor 670 (Cat.65-0840-85, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions (Thermo Fisher Scientific), CD8⁺ T cells were co-cultured with MDSCs pretreated with or without SFN (10 µM). After 48 hours, the cells were collected for flow cytometry analysis.
B16-OVA cells were co-cultured with MDSCs and CD8⁺ T cells from OT-1 mouse spleens. After incubation at 37°C for 24 hours, the adherent B16-OVA cells were digested and labeled with annexin V and PI. Apoptosis was detected using flow cytometry.

Liu J., Chen H., Guo C., Li J., Li M., Zhao M., Fu Z., Zhang Z., Li F., Zhao X., Yang L., Wang L., Lv Q, & Zhang Y. (2024). Sulforaphane activates CD8+ T cells antitumor response through IL-12RB2/MMP3/FasL-induced MDSCs apoptosis’. Journal for Immunotherapy of Cancer, 12(1), e007983.

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CAR-T Cell Cytotoxicity Assay

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Assays were performed on CAR-T cell culture day 3. L1.2 cells were stained with Tag-it Violet Proliferation and Cell Tracking Dye (Biolegend, San Diego, CA, USA), mRobo4-expressing L1.2 cells, and hRobo4-expressing L1.2 cells with Cell Proliferation Dye eFluor 670 (Thermo Fisher Scientific, Waltham, MA, USA). These were used as target cells and cocultured for 18 h with 1 × 10⁵ cells each and CAR-T cells at a concentration suitable for the effector/target ratio of each well. After 18 h, Count Bright Absolute Counting Beads (Thermo Fisher Scientific, Waltham, MA, USA) were added to unify sample analysis volumes, 7-AAD Viability Staining Solution (BioLegend, San Diego, CA, USA) was added to stain dead cells, and analysis was performed using a flow cytometer. Each analysis was completed when 1000 Count Bright Absolute Counting Beads were detected in each sample. The ratio (R) of the number of mRobo4/hRobo4-expressing L1.2 cells to the number of living L1.2 cells was calculated for each well, and the cytotoxic activity was calculated from the following formula.
Cytotoxicity (%) = R (control well) − R (test well)/R (control well) × 100
Control wells were seeded with target cells only, whereas test wells were seeded with both target and effector cells.

Hirobe S., Nagai S., Tachibana M, & Okada N. (2022). Binding and Efficacy of Anti-Robo4 CAR-T Cells against Solid Tumors. Biomedicines, 10(6), 1273.

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VEGFR2-Specific Cytotoxicity Assay

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VEGFR2⁻ target cells (EL4 and L1.2 cells) were labeled with Tag-It Violet Proliferation Cell Tracking Dye (Biolegend), and VEGFR2⁺ target cells (mVEGFR2⁺ EL4 cells and hVEGFR2⁺ L1.2 cells) were labeled with Cell Proliferation Dye eFluor 670 (Thermo Fisher Scientific). Mouse CAR-T cells four days after Rv transduction or human CAR-T cells 24 h after electroporation, VEGFR2⁻ target cells, and VEGFR2⁺ target cells were co-cultured at the indicated effector cells-to-VEGFR2⁺ target cells (E/T) ratios. After 18 h, CountBright Absolute Cell Counting Beads (Thermo Fisher Scientific) and 7-AAD Viability Staining Solution (Biolegend) were added to the reaction wells, and the number of each target cell was analyzed using flow cytometry until 1000 beads were detected. Cytotoxicity was calculated using the following formula: percentage of antigen-specific lysis = [(VEGFR2⁺ target cells/VEGFR2⁻ target cells ratio in non-effector cell’s well) − (VEGFR2⁺ target cells/VEGFR2⁻ target cells ratio in effector cell’s well)]/[VEGFR2⁺ target cells/VEGFR2⁻ target cells ratio in non-effector cell’s well] × 100.

Fujiwara K., Tsunei A., Kusabuka H., Ogaki E., Tachibana M, & Okada N. (2020). Hinge and Transmembrane Domains of Chimeric Antigen Receptor Regulate Receptor Expression and Signaling Threshold. Cells, 9(5), 1182.

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FACS-based ADCC Assay for HIV-1

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Cytotoxicity Assay for VEGFR2+ L1.2 Cells

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L1.2 cells were labeled with Tag-It Violet Proliferation Cell Tracking Dye (Biolegend, San Diego, CA, USA), and VEGFR2⁺ L1.2 cells were labeled with Cell Proliferation Dye eFluor 670 (Thermo Fisher Scientific, Waltham, MA, USA). mRNA-transfected CAR-T cells 24 h after EP, L1.2 cells, and VEGFR2⁺ L1.2 cells were co-cultured at the indicated effector cells-to-VEGFR2⁺ L1.2 cells (E/T) ratios. After 18 h, CountBright Absolute Cell Counting Beads (Thermo Fisher Scientific, Waltham, MA, USA) and 7-AAD Viability Staining Solution (Biolegend, San Diego, CA, USA) were added to the reaction wells, and the number of each target cell was analyzed using flow cytometry until 1000 beads were detected. Cytotoxicity was calculated using the following formula: percentage of VEGFR2-specific lysis = [(VEGFR2⁺ L1.2 cells/L1.2 cells ratio in the non-effector cells’ well) − (VEGFR2⁺ L1.2 cells/L1.2 cells ratio in the effector cells’ well)]/[VEGFR2⁺ L1.2 cells/L1.2 cells ratio in the non-effector cells’ well] × 100.

Fujiwara K., Sasawatari S., Nakai S., Imaeda K., Nagai S., Matsuno Y., Hatanaka K., Hatanaka Y., Takenaka S, & Okada N. (2020). Predicting the Efficacy and Safety of TACTICs (Tumor Angiogenesis-Specific CAR-T Cells Impacting Cancers) Therapy for Soft Tissue Sarcoma Patients. Cancers, 12(10), 2735.

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Thymic Cell Isolation and Labeling

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Thymuses from C57BL/6, lpr, or OT1 mice were harvested, cleaned of connective tissue, mechanically dissociated with a syringe plunger in 6 cm dishes (Alpha, Taiwan) in PBS, and filtered through 70 μm Nylon mesh (Small Parts). For experiments involving overlaying labeled cells on thymic slices, 10⁷ cells were transferred in a 15 mL conical tube (Jet Biofil), washed with PBS, and resuspended in PBS containing 2 μM Cell Proliferation Dye eFluor450 (ThermoFisher) or 1 μM Cell Proliferation Dye eFluor670 (ThermoFisher). The cell suspension was incubated at 37°C for 15 min, washed with complete DMEM (cDMEM) consisting of high glucose DMEM, 10% fetal bovine serum, 1% L-Glutamine, 1% Penicillin/Streptomycin, and 0.1% 2-mercaptoethanol (all from Gibco) and resuspended in cDMEM at a density of 5 × 10⁵/10 μL. The WT cells labeled with eFluor670 and the lpr cells labeled with eFluor450 were mixed at a 1:1 ratio and used for overlaying lpr thymic slices for experiments involving sFasL stimulation. Alternatively, WT cells labeled with eFluor670 and OT1 cells labeled with eFluor450 were mixed and used for overlaying thymic slices for negative selection experiments.

Chu C.F., Feng H.K., Sun K.H., Hsu C.L, & Dzhagalov I.L. (2020). Examination of Fas-Induced Apoptosis of Murine Thymocytes in Thymic Tissue Slices Reveals That Fas Is Dispensable for Negative Selection. Frontiers in Cell and Developmental Biology, 8, 586807.

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