Flow cytometric analysis of RL-7 (blue) and Jurkat (green) cells using Helios (D8W4X) XP® Rabbit mAb (PE-Cy7® Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE-Cy7® Conjugate) (dashed lines).
This Cell Signaling Technology antibody is conjugated to phycoerythrin in combination with cyanine 7 (PE-Cy7®) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Helios (D8W4X) XP® Rabbit mAb #42427.
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 for a full 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 June 2020
Protocol Id: 407
Helios (D8W4X) XP® Rabbit mAb (PE-Cy7® Conjugate) recognizes endogenous levels of total Helios protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly73 of human Helios protein.
Helios (Ikaros family zinc finger 2, IZKF2) is an Ikaros family transcription factor composed of several zinc fingers that mediate DNA binding and homodimerization or heterodimerization with other Ikaros family proteins (1,2). In the hematopoietic system, Helios expression is restricted to T cells and early hematopoietic progenitors (1,2). In regulatory T cells, expression of Helios contributes to an anergic phenotype by binding to the IL-2 promoter and suppressing IL-2 transcription (3). In addition, alteration of the corresponding Helios gene IZKF2 is one hallmark of low-hypodiploid acute lymphoblastic leukemia (4).
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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.
Cy and CyDye are registered trademarks of GE Healthcare.