Assessment of Cornea Viability by Confocal Laser Scanning Microscopy and MTT Assay

Imbert, D; Cullander, Christopher

doi:10.1097/00003226-199711000-00011

Cited by 28 publications

(10 citation statements)

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“…Although calcein itself is a rather inert fluorescein analogue and it has even been used even as a vital dye [1,10,16], its acetoxymethyl derivative can be toxic at high concentrations in tumor cells [17,19]. Nevertheless, if calcein-AM were found to affect cell viability, other dyes are available such as BCECF or Fura-2 used at their isosbestic wavelengths [2].…”

Section: Discussionmentioning

confidence: 99%

Measurement of sugar transport in single living cells

Barros

1999

Pfl�gers Archiv European Journal of Physiology

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A fluorometric method that allows repeatable measurement of sugar transport rates and parameters in single living cells is presented. Intracellular sugar concentrations were estimated in real time from changes in cell volume that occur secondary to permeation of sugars across the plasma membrane. In turn, the cell volume changes were estimated from variations of intracellular calcein fluorescence measured by confocal microscopy. Using HeLa cells, the assay allowed reproducible measurement of the uptake and exit of D-galactose and 3-O-methyl-D-glucose. The rate of zero-trans uptake (i.e. at an intracellular concentration of zero) of galactose at an extracellular concentration of 200 mM was 0.34+/-0.05 mM/s (n=8). Apparent Vmax and Km for galactose exit were 0.32+/-0.05 mM/s (n=9) and 30+/-7.2 mM (n=9), respectively. The apparent affinity of infinite-trans (i.e. at a very high intracellular concentration) uptake of 3-O-methyl-D-glucose was 3.8+/-0.47 mM (n=8). Galactose uptake was 93+/-8% (n=8) inhibited in the presence of 50 microM phloretin, whereas galactose exit was 96+/-6% (n=5) trans-inhibited by 100 mM 4,6-ethylidine-D-glucose. This technique may help to characterize sugar transport in freshly isolated cells, co-cultures and heterogeneous cell explants. It may also allow available cell microinjection technology to be used to study the regulation of sugar transporters' intrinsic activity. In principle, similar approaches might also be applied in functional studies of other transporters for which non-metabolized substrates are available.

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Section: Discussionmentioning

confidence: 99%

Measurement of sugar transport in single living cells

Barros

1999

Pfl�gers Archiv European Journal of Physiology

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“…Therefore, it seems appropriate that the viability of excised buccal tissue be assessed, as this may affect the observed permeability results. Consequently, the viability of porcine buccal tissue was determined using histological evaluation and an MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) biochemical assay that has previously been used in assessing the viability of excised buccal mucosa and cornea 13,14…”

Section: Introductionmentioning

confidence: 99%

The Effect of Various In Vitro Conditions on the Permeability Characteristics of the Buccal Mucosa

Nicolazzo

Reed

Finnin

2003

Journal of Pharmaceutical Sciences

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“…CLM with vital cell staining is recognized as an accurate and sensitive method of determining cell viability. 1,[5][6][7][8][9][10][11][12][13] This study describes the use of CLM in conjunction with vital cell staining (Live/Dead Viability/Cytotoxicity Kit [L-3224], Molecular Probes, Eugene, OR). This technique uses calcein-AM (acetoxymethylester) to label live cells and ethidium homodimer-1 (EthD-1) for dead cells.…”

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confidence: 99%