Flow cytometric analysis of HeLa cells (blue) and KARPAS-299 cells (green) using TNFRSF8/CD30 (E7E4D) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 555 Conjugate) #3969 (dashed lines). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye 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 TNFRSF8/CD30 (E7E4D) XP® Rabbit mAb #25114.
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.
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: 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 live cell staining is desired, proceed to Section C.
NOTE: Count cells using a hemocytometer or alternative method.
posted January 2009
revised June 2020
Protocol Id: 180
TNFRSF8/CD30 (E7E4D) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) recognizes endogenous levels of total TNFRSF8/CD30 protein.
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the extracellular domain of human TNFRSF8/CD30 protein.
TNFRSF8/CD30 is a type-I transmembrane glycoprotein that is a member of the TNFR superfamily. CD30 is synthesized as a precursor protein that undergoes extensive posttranslational modification before becoming embedded in the plasma membrane as a 120-kDa transmembrane protein (1,2). The expression of CD30 is upregulated in activated T-cells and may trigger costimulatory signaling pathways upon its engagement (3,4). While its expression is normally restricted to subsets of activated T-cells and B-cells, CD30 expression is robustly upregulated in hematologic malignancies, such as Hodgkin’s lymphoma (HL), anaplastic large cell lymphoma (ALCL), and adult T-cell leukemia, thus making it an attractive target for therapeutic intervention (5,6). Research studies have suggested that in certain disease contexts, CD30 recruits TRAF2 and TRAF5 adaptor proteins to drive NF-kappa B activation, aberrant cell growth, and cytokine production (7-9). CD30 signaling is also regulated by TACE-dependent proteolytic cleavage of its ectodomain, which results in reduced CD30L-dependent activation of CD30+ cells (10, 11).
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