Detection of terminal deoxynucleotidyl transferase by flow cytometry: A three color method

Joseph M. Horvatinovich, Sara D. Sparks, Michael J. Borowitz

Research output: Contribution to journalArticle

Abstract

This short communication describes a three‐color flow cytometric analysis for terminal deoxynucleotidyl transferase (TdT). The method combines CD45‐gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three‐color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti‐TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three‐color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expressionof anti‐TdT FITC and a PE conjugated MoAb identifies TdT useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45‐defined blast populations. © 1994 Wiley‐Liss, Inc.

Original languageEnglish (US)
Pages (from-to)228-230
Number of pages3
JournalCytometry
Volume18
Issue number4
DOIs
StatePublished - Dec 15 1994

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Keywords

  • CD45 perCP
  • FACS lysing solution
  • Permeabilization
  • leukemia immunophenotyping

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

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