Efficient fixation procedure of human leukemia cells in sulforhodamine B cytotoxicity assay

Hwan Mook Kim, Sang Bae Han, Moon Soon Kim, Jong Seong Kang, Goo Taeg Oh, Dong Ho Hong

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

Abstract

The fixation procedures in sulforhodamine B (SRB) assay for human leukemia cells were modified to produce more reliable results. It was found that the concentration of the fixative agent, trichloroacetic acid (TCA), was critical in the selective fixation of cellular protein. While a TCA solution of 80% fixed both cells and serum proteins, a 50% solution fixed only cells with a very low interference of the serum proteins. Accordingly, we selected 50% TCA as a fixative agent which made the final absorbance of the SRB assay to be exactly matched to the cell density with a small deviation and a low background. Besides the change of TCA concentration, a precentifugation of microplate just before fixation also improved the previous assay procedures in the two points of view. The 2-h standing step was simply substituted for only 1 min of centrifugation. Both the rapid and slow application of TCA solution in fixation produced the same extents of fixation. In an actual application, these two kinds of modifications in the previous SRB assay procedure were also proved to be effective in the determination of cytotoxicities of doxorubicin by using human leukemias.

Original languageEnglish
Pages (from-to)163-169
Number of pages7
JournalJournal of Pharmacological and Toxicological Methods
Volume36
Issue number3
DOIs
StatePublished - Nov 1996

Keywords

  • Cytotoxicity
  • Leukemias
  • SRB assay
  • TCA

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