Efluor 450 cell proliferation dye cpd

Manufactured by Thermo Fisher Scientific

The EFluor 450 cell proliferation dye (CPD) is a cell labeling reagent used for tracking cell division. It binds to cellular proteins, providing a fluorescent signal that is distributed equally between daughter cells during cell division.

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Lab products found in correlation

Recombinant human siglec 6 fc chimera protein, by R&D Systems (2 mentions) Zombie aqua, by BioLegend (1 mentions) Phosflow perm buffer 3, by BD (1 mentions) Cytofix fixation buffer, by BD (1 mentions) Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Cd11c dcs, by Miltenyi Biotec (1 mentions) Anti cd3ε, by BD (1 mentions) Streptavidin microbeads, by Miltenyi Biotec (1 mentions)

Close spelling variants of this product

EFluor 450 (209 citations) Cell Proliferation Dye eFluor 450 (69 citations) Cell proliferation dye (10 citations) EFluor 450 proliferation dye (8 citations) Cell proliferation dye 450 (2 citations)

5 protocols using efluor 450 cell proliferation dye cpd

Proliferation and Cytotoxicity Assay for CAR T Cells

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CAR-transduced T cells were labeled with 10 μM eFluor 450 cell proliferation dye (CPD, eBioscience) according to the manufacturer’s instructions. 5 × 10⁵ CPD-labeled transduced CAR T cells were plated on 24 well/plates coated with recombinant human Siglec-6/Fc chimera protein (R&D System, Minneapolis, MN). Proliferation of T cells was analyzed by flow cytometry.
CAR T cell cytotoxicity assay was performed as described (20 (link)) with some modifications. Briefly, 2-week expanded, CAR T cells were co-cultured with target cells at the indicated ratios for 4-6 hours and analyzed by flow cytometry. %specific lysis=[(negative target-experimental target)/negative target x100]. The cytotoxic assay on primary CLL cells was performed by mixing a similar number of CAR-transduced T cells and primary CLL cells followed by co-culture for 5hs. Remaining CLL cells in culture were identified by flow cytometry as CD5⁺CD19⁺ cells.

Kovalovsky D., Yoon J.H., Cyr M.G., Simon S., Voynova E., Rader C., Wiestner A., Alejo J., Pittaluga S, & Gress R.E. (2021). Siglec-6 is a Target for Chimeric Antigen Receptor T-cell Treatment of Chronic Lymphocytic Leukemia. Leukemia, 35(9), 2581-2591.

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Proliferation and Cytotoxicity Assay for CAR T Cells

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Splenic B Cell Proliferation Assay

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Purified splenic B cells were labeled with eFluor 450 cell proliferation dye (CPD; eBioscience) according to the manufacturer’s protocol. Cells were cultured for 72 h in full medium in flat-bottom 96-well plates with stimulants as in cell survival assays, before harvesting and analysis on a flow cytometer. Dead cells were excluded by staining with Zombie Aqua (BioLegend). Calibrite beads allowed quantitation of live cells. The proliferation module of FlowJo was used to determine the percentage of cells that had divided at least once based on dilution of the CPD.

Schweighoffer E., Nys J., Vanes L., Smithers N, & Tybulewicz V.L. (2017). TLR4 signals in B lymphocytes are transduced via the B cell antigen receptor and SYK. The Journal of Experimental Medicine, 214(5), 1269-1280.

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T-cell Proliferation and ERK Phosphorylation

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Total PBMCs were stained with Cell Proliferation Dye eFluor 450 (CPD; Affymetrix, eBiosciences) at 20 μm for 10 min, then washed and stimulated with Dynabeads^® Human T‐Activator CD3/CD28 (Life Technologies, Carlsbad, CA). Three days later, the percentage of CPD‐low cells was evaluated within naïve (CD45RA⁺ CCR7⁺ CD27⁺) CD8⁺ T‐cells by flow cytometry. To assess ERK1 and ERK2 phosphorylation, PBMCs were stained with T‐cell differentiation surface markers for 15 min at room temperature and then exposed to Dynabeads^® Human T‐Activator CD3/CD28 (Life Technologies) for 2 min at 37°C. Cells were subsequently washed and fixed in BD Cytofix Fixation Buffer (BD Biosciences) for 10 min at 37°C and then permeabilized with BD Phosflow^™ Perm Buffer III (BD Biosciences) for 20 min at 4°C. After washing, cells were stained intracellularly for 1 h at room temperature using BD Phosflow^™ anti‐human ERK1/2 conjugated to AlexaFluor647 (BD Biosciences) and analyzed by flow cytometry.

Briceño O., Lissina A., Wanke K., Afonso G., von Braun A., Ragon K., Miquel T., Gostick E., Papagno L., Stiasny K., Price D.A., Mallone R., Sauce D., Karrer U, & Appay V. (2015). Reduced naïve CD8+ T‐cell priming efficacy in elderly adults. Aging Cell, 15(1), 14-21.

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Assessing B-cell Antigen Presentation and Suppression

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To assess B-lymphocyte mediated antigen presentation, CD4⁺ T-cells purified from spleens of 8–10 week old NOD BDC2.5 TCR transgenic mice by negative depletion of CD11b⁺, CD11c⁺, TER-119⁺, B220⁺, and CD8⁺ cells using streptavidin microbeads (Miltenyi) were labeled with 2 μM Cell Proliferation Dye eFluor450 (CPD) (eBiosciences) according to the manufacturer’s protocol. Labeled CD4⁺ T-cells were cultured in the presence of 1 μM BDC2.5 mimotope (AHHPIWARMDA, Mimotopes) with or without sorted B-lymphocytes for 3 days. T-cell proliferation was assessed by flow cytometric analyses of CPD dilution as a measure of antigen presentation. In another experiment, microbead purified CD11c⁺ DCs (Miltenyi) were used as an APC source. For B-lymphocyte mediated suppression, CD4⁺ CD25⁻ T-cells purified from spleens of 8–10 week old NOD mice were labeled with 2 μM CPD. Sorted B-lymphocytes and labeled T-cells stimulated with plate-bound 5 μg/mL anti-CD3ε (BD Biosciences) were co-cultured with 0 or 10μg/ml LPS (Sigma-Aldrich) for 3 days. T-cell proliferation (CPD dilution) was assessed by flow cytometry and data analyzed using FlowJo software. Percent suppression was calculated relative to the mean Proliferation Index of anti-CD3 stimulated T-cells co-cultured with B-lymphocytes in medium without LPS.

Wang Q., Racine J.J., Ratiu J.J., Wang S., Ettinger R., Wasserfall C., Atkinson M.A, & Serreze D.V. (2017). Transient BAFF blockade inhibits type 1 diabetes development in NOD mice by enriching immunoregulatory B-lymphocytes sensitive to deletion by anti-CD20 co-therapy,. Journal of immunology (Baltimore, Md. : 1950), 199(11), 3757-3770.

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