Invitrogen Antibodies
Cell Proliferation Dye eFluor™ 450
65-0842-85
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mTORC1 activity very critical for the generation of effector T cells but also controls cell proliferation through metabolic programming. Our lab studies the asymmetric division of mTORC1 activity during CD8 T cell differentiation. We used this dye to monitor cell proliferation via flow cytometry during an in vivo immune response. We were interested in assessing early proliferation within 48hrs of an immune response. This dye is quite reliable for monitoring at least six to seven divisions without inducing cell death during the labeling procedure.
Cell proliferation via flow cytometry
Murine CD8 OT1 T cells
We reconstituted the dye as mentioned. We obtained single cell suspension of the T cells in warm PBS. The cells were adjusted to 10e6/mL in PBS then stained with 1:1000 of the ef450 dye for 10 minutes at 37 deg. The reaction was quenched with cold media on ice for 5 minutes then washed once before downstream application.
Use warm PBS and consistent cell count for consistency between experiments
We were able to assess multiple cell divisions from the adoptively transferred cells.
27064374
N/A
Very consistent staining and viability of cells
None at all
We no longer use CFSE to monitor cell proliferation since viability and consistency were always an issue. eF450 beats CFSE!