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E450 cell proliferation dye

Manufactured by Thermo Fisher Scientific
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The E450 cell proliferation dye is a fluorescent labeling reagent designed for the measurement of cell proliferation. It functions by covalently binding to cellular proteins, allowing for the tracking of cell division through successive generations.

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Lab products found in correlation

Ovalbumin (ova), by STEMCELL (1 mentions) Ovalbumin (ova), by Thermo Fisher Scientific (1 mentions) Easysep mouse cd8 t cell enrichment kit, by STEMCELL (1 mentions) Vybrant cfda se cell tracer kit, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions)

5 protocols using e450 cell proliferation dye

1

Antigen Presentation by OPCs to CD8 T cells

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Assay was performed as described with minor modifications[40 (link)]. Cultured OPCs were plated in a 96-well plate at 20,000 cells per well. The next day, OPCs were treated with 10ng/mL IFNγ for 12h. OPCs were then pre-treated with 300nM GST-RAP or GST (vehicle) for 1h in IFNγ-containing OPC media. Next, OPCs were incubated with 500μg/mL of OVA (77120, Thermo Fisher) in IFNγ-containing OPC media in the presence of GST-RAP or GST for 8h. During the 8h OVA incubation, OT-I CD8 T cells were isolated (19853, Stem Cell Technologies) from spleen and lymph nodes and labeled with Cell Proliferation Dye e450 (65–0842-85, eBioscience). OPCs were then washed with PBS and 160,000 OT-I CD8 T cells were added in a 50/50 mix of RPMI complete and OPC media. OT-I CD8 T cell proliferation was examined using flow cytometry after 48h of coculture.
Fernández-Castañeda A., Chappell M.S., Rosen D.A., Seki S.M., Beiter R.M., Johanson D.M., Liskey D., Farber E., Onengut-Gumuscu S., Overall C., Dupree J.L, & Gaultier A. (2019). The active contribution of OPCs to neuroinflammation is mediated by LRP1. Acta neuropathologica, 139(2), 365-382.
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2

Adoptive Transfer of Antigen-Specific CD8+ T Cells

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CD8+ T cells were purified from P14 (GP33–41-specific TCR) and TNP4 (NP396–404-specific TCR) transgenic mice using the EasySep mouse CD8+ T-cell enrichment kit (Stemcell Technologies). Purified CD8+ T cells (1.5 × 106) from P14 mice were stained with CFSE (5 μM) (Vybrant CFDA SE Cell Tracer Kit; Molecular Probes), and cells from TNP4 mice (1.5 × 106) with Cell Proliferation Dye e450 (10 μM) (eBioscience). We adoptively transferred 1.5 × 106 CD8+ T cells from P14 and TNP4 mice by IV route into TTR-NP and B6 control mice. The next day, the mice were infected with 200 pfu of LCMV-Arm IP, and they were killed 4 days later.
Alternatively, 1.5 × 106 CD8+ T cells from P14 and TNP4 mice (Stemcell Technologies) were adoptively transferred IV into RAG-NP and RAG control mice. The mice were killed 7 days later. Isolated lymphocytes from the spleen and liver were stained (CD3, CD4, CD8, and 7-AAD), and the proliferation of CFSE and e450-labeled cells was analyzed by flow cytometry on a BD LSRFortessa (BD Biosciences) and using the FlowJo software (Tree Star).
Lapierre P., Janelle V., Langlois M.P., Tarrab E., Charpentier T, & Lamarre A. (2015). Expression of Viral Antigen by the Liver Leads to Chronic Infection Through the Generation of Regulatory T Cells. Cellular and Molecular Gastroenterology and Hepatology, 1(3), 325-341.e1.
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3

Adoptive Transfer of Antigen-Specific B Cells

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For adoptive transfer experiments, B cells purified from spleens of CD45.1 WT mice and CD45.2 MD4 mice were mixed at a 1:1 ratio, stained with e450 cell proliferation dye (ThermoFisher) according to the manufacturer’s recommendations, and resuspended in PBS. 200 μl PBS containing 4.5 × 106 e450-stained B cells were transferred into a CD45.2 WT mouse via tail vein injection. 24 h post-transfer mice were injected with 200 μl PBS alone or PBS supplemented with 95-100 μg/mouse ODN1826 and/or 100 μg/mouse HEL. 24 h post-stimulation spleens were harvested and splenocytes were used in downstream assays.
Akkaya M., Traba J., Roesler A.S., Miozzo P., Akkaya B., Theall B.P., Sohn H., Pena M., Smelkinson M., Kabat J., Dahlstrom E., Dorward D.W., Skinner J., Sack M.N, & Pierce S.K. (2018). Second signals rescue B cells from activation-induced mitochondrial dysfunction and death. Nature immunology, 19(8), 871-884.
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4

Adoptive Transfer of OT-II T Cells

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For adoptive transfer experiments, naïve CD4+ T cells purified from spleens of CD45.1+CD45.2+ WT mice and CD45.2+ OT-II transgenic mice were mixed at a 1:1 ratio, stained with e450 cell proliferation dye (ThermoFisher) according to the manufacturer’s recommendations, and resuspended in PBS. 200 μl PBS containing 2 × 106 e450-stained CD4+ T cells were transferred into a CD45.2 WT mouse i.v. via tail vein injection. 24 h post T cell transfer, mice were injected i.v. with 100 μl PBS containing 7.5 X105 dendritic cells (DCs) previously loaded with either OVA(323–339) or LCMV GP(61–80) peptides. Four days post DC transfer, mice were euthanized and spleens were harvested.
Akkaya B., Roesler A.S., Miozzo P., Theall B.P., Souz J.A., Smelkinson M.G., Kabat J., Traba J., Sack M.N., Brzostowski J.A., Pena M., Dorward D.W., Pierce S.K, & Akkaya M. (2018). Increased mitochondrial biogenesis and ROS production accompany prolonged CD4+ T cell activation. Journal of immunology (Baltimore, Md. : 1950), 201(11), 3294-3306.
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5

Adoptive Transfer of Antigen-Specific B Cells

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For adoptive transfer experiments, B cells purified from spleens of CD45.1 WT mice and CD45.2 MD4 mice were mixed at a 1:1 ratio, stained with e450 cell proliferation dye (ThermoFisher) according to the manufacturer’s recommendations, and resuspended in PBS. 200 μl PBS containing 4.5 × 106 e450-stained B cells were transferred into a CD45.2 WT mouse via tail vein injection. 24 h post-transfer mice were injected with 200 μl PBS alone or PBS supplemented with 95-100 μg/mouse ODN1826 and/or 100 μg/mouse HEL. 24 h post-stimulation spleens were harvested and splenocytes were used in downstream assays.
Akkaya M., Traba J., Roesler A.S., Miozzo P., Akkaya B., Theall B.P., Sohn H., Pena M., Smelkinson M., Kabat J., Dahlstrom E., Dorward D.W., Skinner J., Sack M.N, & Pierce S.K. (2018). Second signals rescue B cells from activation-induced mitochondrial dysfunction and death. Nature immunology, 19(8), 871-884.
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