Flow cytometric analysis of Jurkat cells (blue) and IGROV-1 cells (green) using FoxO1 (C29H4) Rabbit mAb (Pacific Blue™ Conjugate) (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Pacific Blue™ Conjugate) #9078 (dashed lines).
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This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for direct flow cytometric analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated antibody FoxO1 (C29H4) Rabbit mAb #2880.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised August 2019
Protocol Id: 407
FoxO1 (C29H4) Rabbit mAb (Pacific Blue™ Conjugate) detects endogenous levels of total FoxO1 protein. The antibody does not detect exogenously expressed family members FoxO3a or FoxO4.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a GST-fusion protein corresponding to carboxy-terminal residues of human FoxO1.
The Forkhead family of transcription factors is involved in tumorigenesis of rhabdomyosarcoma and acute leukemias (1-3). Within the family, three members (FoxO1, FoxO4, and FoxO3a) have sequence similarity to the nematode orthologue DAF-16, which mediates signaling via a pathway involving IGFR1, PI3K, and Akt (4-6). Active forkhead members act as tumor suppressors by promoting cell cycle arrest and apoptosis. Increased expression of any FoxO member results in the activation of the cell cycle inhibitor p27 Kip1. Forkhead transcription factors also play a part in TGF-β-mediated upregulation of p21 Cip1, a process negatively regulated through PI3K (7). Increased proliferation results when forkhead transcription factors are inactivated through phosphorylation by Akt at Thr24, Ser256, and Ser319, which results in nuclear export and inhibition of transcription factor activity (8). Forkhead transcription factors can also be inhibited by the deacetylase sirtuin (SirT1) (9).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Pacific Blue is a trademark of Molecular Probes, Inc.
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