Quantitation of two histochemical markers in the same extract using chemiluminescent substrates

K. L. O'Connor, L. A. Culp

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Factors that influence early events of primary tumor development have been cumbersome to evaluate because of the need to either wait for tumor palpability after experimental manipulation or use of radiolabel to evaluate cell clearance. To facilitate these and similar analyses of cells in vivo, new methods are described that utilize histochemical marker genes to quantitate tumor cell number in a target tissue through the use of luminescent, enzymatic assays for these gene products. 3T3 Cells transfected with either human placental alkaline phosphatase or bacterial lacZ genes were injected subcutaneously into athymic nude mice. Using luminescent substrates designed for marker gene enzymes, extracts from homogenized tumor cell- bearing skins were assayed for the corresponding marker enzyme activities, which were optimized for recovery from skin extracts and correlated to cell number. The homogenization buffer used for these assays was designed to accommodate the optimal and simultaneous recovery of cytosolic β- galactosidase and membrane-linked alkaline phosphatase from the skin, as well as from cultured cells. These assays provide an inexpensive, sensitive method for quantitatively monitoring the fate of cells genetically tagged with marker genes in various in vivo environments.

Original languageEnglish
Pages (from-to)502-504+506+508
JournalBioTechniques
Volume17
Issue number3
StatePublished - 1994

Funding

FundersFunder number
National Childhood Cancer Registry – National Cancer InstituteR01CA027755

    ASJC Scopus subject areas

    • Biotechnology
    • General Biochemistry, Genetics and Molecular Biology

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