Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.

A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)

Caudalization of neural fate by tissue recombination and bFGF.

In order to study anteroposterior neural patterning in Xenopus embryos, we have developed a novel assay using explants and tissue recombinants of early neural plate. We show, by using region-specific neural markers and lineage tracing, that posterior axial tissue induces midbrain and hindbrain fates from prospective forebrain. The growth factor bFGF mimics the effect of the posterior dorsal explant in that it (i) induces forebrain to express hindbrain markers, (ii) induces prospective hindbrain explants to make spinal cord, but not forebrain and midbrain, and (iii) induces posterior neural fate in ectodermal explants neuralized by the dominant negative activin receptor and follistatin without mesoderm induction. The competence of forebrain explants to respond to both posterior axial explants and bFGF is lost by neural groove stages. These findings demonstrate that posterior neural fate can be derived from anterior neural tissue, and identify a novel activity for the growth factor bFGF in neural patterning. Our observations suggest that full anteroposterior neural patterning may be achieved by caudalization of prospective anterior neural fate in the vertebrate embryo.

An eye banking program for selecting donor corneas for surgical distribution.

Since fewer donated corneas have become available for surgery, we sought to chart the reasons to exclude them for surgical use over time. Those excluded from surgical use (1991-1994) were plotted using an algorithm based on the reasons for exclusion. Four general categories (universal contraindications [UC], national/local medical criteria [NLMC], serology, and morphology) yielded 13 possible areas. UC and NLMC exclusions for 1993-1994 were higher compared with 1991 and 1992 (p < 0.001). The proportion of corneas excluded for serological reasons decreased (p < 0.001) from 1991 to 1994. Exclusions due to morphology remained the same for all 4 years (p = NS). NLMC eliminate older donors but also exclude younger donors before the tissue reaches the eye bank (p > 0.001). Three of four of the youngest tissues ( < 30 years) are used for surgery, whereas one of five of the oldest ( > 70 years) is used. A quality control algorithm provides a heuristic and logical paradigm for noting changes from year to year. Heightened regulation has counteracted many gains in corneal donation fostered by favorable laws.

Cardiac myosin heavy chain expression during heart development in Xenopus laevis.

Muscle-specific gene expression in the heart during Xenopus development was investigated using reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization to detect transcripts of the gene for the cardiac myosin heavy chain (CMHC). RT-PCR analysis determined that CMHC transcripts are present in the cardiac mesoderm at state 13, demonstrating that muscle-specific gene expression in the primitive myocardium has begun by the early neurula stage, approximately 30 h before the heart beat begins. Xenopus, therefore, is similar to amniotes and mammals in that cardiac precursor cells begin to express muscle-specific gene transcripts soon after commitment to the cardiac myocyte lineage. The earliest CMHC gene transcripts can be detected in the heart using whole-mount in situ hybridization is early tailbud stage 28, which coincides with the onset of heart tube morphogenesis. CMHC gene expression was also detected in skeletal muscle: RT-PCR analysis determined that CMHC transcripts are transiently expressed in the somite during the initial phases of skeletal muscle differentiation. Furthermore, CMHC mRNAs are expressed in a subset of head muscles of the feeding tadpole. CMHC gene expression is induced in ectodermal cells of the animal cap in blastula-stage embryos injected with synthetic MyoD or Myf5 RNA, suggesting that the CMHC gene contains regulatory elements that are responsive to the activity of those skeletal-muscle-specific transcription factors.