A kinetic study on the enzymatic hydrolysis of fluoresceindiacetate and fluorescein-di-β-d-galactopyranoside

Hofmann, Jürgen; Sernetz, M.

doi:10.1016/0003-2697(83)90151-3

Cited by 64 publications

(57 citation statements)

References 7 publications

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“…With the FDG loading protocol presented in this paper, cells are loaded to -3 x 1O-4 M FDG (data not shown). This is adequate to provide a concentration of substrate in excess of the KM, given a Km of 17 x 10-6 M of /3-galactosidase LacZ for FDG (19), so that the initial rate of hydrolysis is not dependent upon substrate concentration.…”

Section: Discussionmentioning

confidence: 99%

“…We have not measured the lowest number of 8-galactosidase molecules detectable; however, based on published turnover rates (19,20) estimated at 40C to be 0.75 or 7 substrate molecules per sec, and if the enzyme has free access to the substrate, one enzyme molecule per cell can theoretically be detected by FACS at 40C in some 30-180 min. Because of the high sensitivity of this enzymatically amplified assay, we expect that it will be useful to quantify expression from weak promoters or to rapidly assess gene activation kinetics.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

Nolan

Fiering

Nicolas

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

457

253

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We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli fi-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescenceactivated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for 13-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of f-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescenceactivated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of (3-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.The Escherichia coli lacZ gene has previously been adapted for use in mammalian cells and has shown utility as a marker for the expression of chimeric genes (1, 2). In these previous studies, the chromogenic f3-galactoside analog o-nitrophenyl /3-D-galactopyranoside (ONPG) was used as an alternative to the chloramphenicol acetyltransferase assay (3) to quantitate gene expression from heterologous promoters fused 5' proximal to the lacZ structural gene. However, in both the chromogenic LacZ and radioactive chloramphenicol acetyltransferase assays, extracts of bulk populations of cells are used to quantitate an average promoter strength/enzyme activity and, thus, do not give any indication of the heterogeneous expression patterns that could exist within complex cell populations. These other assays also do not provide one with a means to select viable cells expressing known quantities of gene product.lacZ has also proven effective as a marker in the genetic tagging of cell lineage founders and subsequent tracing of progeny in tissue sections using a cytochemical stain for expression of LacZ (4, 5). Using recombinant, replicationdefective Moloney murine leukemia viruses (Mo-MuLV) in which the expression of lacZ is permitted from an internal simian virus 40 (SV40) promoter, Sanes et al. (4) Here, we use the f3-galactoside analog fluorescein di-,B-Dgalactopyranoside (FDG) (11) in a protocol, termed " 'ACS-FDG," that sensitively distinguishes LacZ' cells from LacZ-cells and allows time-dependent fluorescenceactivated cell analysis and sorting. FDG is cleaved by 03-galactosidase in LacZ' cells to yield fluorescein, which can be detected by FACS. We find that the kinetics of fluorescence accumulation within LacZ' cells, after FDG substrate loading, shows a linear dependence over time until maxima are approached because of...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

Nolan

Fiering

Nicolas

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

457

253

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“…This carboxyfluorescein derivative is lipophilic and can therefore cross the plasma membrane into the cytosol, where nonspecific intracellular esterases cleave the acetoxymethyl ester bonds releasing the free form of the fluorophore that is well retained within the cell (Grimes et al, 1982 ;Hofmann and Sernetz, 1983 ;Rotman and Papermaster, 1966). Exposure of cells having incorporated BCECF-AM to light at the appropriate wavelength leads to cellular photoablation.…”

Section: Discussionmentioning

confidence: 99%

“…Several photosensitizers have been advocated to treat localized pathologies in distinct clinical entities (Dougherty and Marcus, 1992 ;Hill et al, 1997 ;Weissgold et al, 1996 ;Moshfeghi et al, 1995 ;Cox et al, 1997 ;Smyth et al, 1993 ;Dacheux and Guidry, 1995). The objective of this study was to evaluate the effect of cellular photoablation mediated by (Grimes et al, 1982 ;Hofmann and Sernetz, 1983 ;Rotman and Papermaster, 1966). Exposure of cells having incorporated BCECF-AM to light at the appropriate wavelength leads to cellular photoablation via the formation of reactive oxygen intermediates and free radicals (Cohan, Hadley and Kater, 1983 ;Dulan, Zajic and Schacht, 1989 ;Miller and Selverstone, 1979 ;Moreno, Lutz and Bessis, 1969).…”

Section: Introductionmentioning

confidence: 99%

Cellular Photoablation to Control Postoperative Fibrosis in Filtration Surgery: in vitro Studies

Grisanti

Gralla

Maurer

et al. 2000

Experimental Eye Research

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“…Intracellularly, BCECF-AM is activated upon cleavage by intracellular esterases, thereby rendered fluorescent and membrane impermeable. [4][5][6] After additional illumination with diffuse blue light at the appropriate wavelength (λ = 450-490 nm, intensity ∼51.9 × 1000 cd/m 2 ), intracellular carboxyfluorescein then exerts the photo-oxidative effect by producing free oxygen radicals (cytodestructive oxidative stress, photooxidative reactions of type I and II), finally leading to the death of the cell.…”

Section: Patientsmentioning

confidence: 99%

Photodynamic Modulation of Wound Healing in Glaucoma Filtration Surgery

Grisanti

2012

Handbook of Biophotonics

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Aim: To report a clinical pilot study investigating photodynamic therapy (PDT) in combination with glaucoma filtration surgery. BCECF-AM was used as the photosensitising substance. The clinical safety and tolerability of BCECF-AM, and its efficacy in controlling postoperative intraocular pressure (IOP) were assessed. Methods: Before trabeculectomy (TE), 42 consecutive eyes of 36 glaucoma patients received one subconjunctival injection of 80 µg BCECF-AM (2,7,-bis-(2-carboxyethyl) -5-(and-6) -carboxy-fluorescein, acetoxymethyl-ester) followed by an intraoperative illumination with blue light (λ = 450-490 nm) for 8 minutes. Antifibrotic efficacy was established as postoperative IOP reduction of >20% and/or an IOP constantly < 21 mm Hg without antiglaucomatous medication. Follow up of the filtering bleb was documented by slit lamp examination. Results: Eyes had mean 1.1 preoperative surgical interventions (filtration and non-filtration glaucoma surgery). Mean preoperative IOP was 31.6 (SD 9.7) mm Hg. Patients were followed for mean 496 days (range 3.5-31.8 months). Of the 42 eyes, 25 eyes had an IOP decreased to 15.8 (3.4) mm Hg without medication (complete success: 59.5%; p<0.001; t test). Seven eyes showed good IOP reduction < 21 mm Hg under topical antiglaucomatous medication (qualified success: 16.7%). 10 eyes failed because of scarring within 2-67 weeks (23.8%). Clinical follow up examinations revealed no local toxicity, no uveitis, and no endophthalmitis. Conclusions: This method is a new approach in modulating postoperative wound healing in human eyes undergoing glaucoma filtration surgery. The data of the first human eyes combining TE with PDT underline the clinical safety of this method and its possible potential to prolong bleb survival. P ostoperative scarring of the filtering bleb is the most crucial factor in determining the short and long term outcome of modern glaucoma filtration surgery. Trabeculectomy (TE) is the preferred operation. However, in conventionally performed trabeculectomy, a large retrospective study has shown a failure rate of up to 30% within 3 months after surgery.

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A kinetic study on the enzymatic hydrolysis of fluoresceindiacetate and fluorescein-di-β-d-galactopyranoside

Cited by 64 publications

References 7 publications

Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

Cellular Photoablation to Control Postoperative Fibrosis in Filtration Surgery: in vitro Studies

Photodynamic Modulation of Wound Healing in Glaucoma Filtration Surgery

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