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A Gaigalas

Publications and source records attributed to A Gaigalas.

5 recordsLinked to original sources
PubMed2004

Quantification of EGFP expression on Molt-4 T cells using calibration standards.

BACKGROUND: Enhanced green fluorescent protein (EGFP) is used extensively to assess gene expression on cells; however, quantification of this expression by flow cytometry has been limited by the unavailability of calibration standards. Thus, we characterized the response of an experimental set of EGFP calibration standards to environmental changes and then quantitate the expression of EGFP, in molecules of equivalent soluble fluorochrome (MESF) units, of a transfected Molt-4 T cell line by flow cytometry. METHODS: Characterization of the EGFP standards: EGFP standards were equilibrated in suspension solutions having a pH range of 5.0-9.0, temperatures of 37-80 degrees C, and osmolalities of 100-600 mOsm/kg. Quantification of EGFP on cells: For transfections, Molt-4 T cells were incubated with two different concentrations (0.2 microg and 0.4 microg) of pEGFP-N2 vector and the EGFP expression was quantified after 48 h by flow cytometry using the EGFP standards and by the cytofluor technique using a standard curve of known EGFP solutions. RESULTS: The fluorescence intensity of the EGFP standards increased from pH 5.0 to 9.0 and remained relatively constant from 37 degrees C to 65 degrees C, and from 100 to 600 mOsm/kg. After transfection, the expression of the populations with high and low EGFP expression averaged 8,098 +/- 584 MESF and 3,808 +/- 375 MESF respectively. No significant differences were observed after comparing the MESF values obtained by flow cytometry and the values obtained by Cytofluor technique (high: 8,791 +/- 492 MESF; low: 4,082 +/- 398 MESF). CONCLUSIONS: Our data demonstrate the feasibility of using calibration standards to quantify EGFP expression on cells. Our results emphasize the importance of monitoring the effects of environmental changes in the fluorescence intensity of both standards and samples when quantifying the expression of EGFP on living cells.

Calibration↗
PubMed1998

Terminology and nomenclature for standardization in quantitative fluorescence cytometry.

Terminology in any field is a complex mix of established conventions, accepted usages, disputed terms, and occasional misnomers. The terminology that has evolved for quantitative fluorescence cytometry (QFCM) is especially multifarious, in part because QFCM encompasses a range from subjective visual assessments to objective photon counts. Thus, while descriptive terms such as "dim" and "bright" are still quite useful, quantitative terms such as "binding capacity" should be used with collective understanding of their exact meanings. This article reviews current usage and proposes definitions that, with refinement from suppliers and users of QFCM technology, can provide the required clarity.

Calibration↗
PubMedDate not supplied

A multistate model for the fluorescence response of R-phycoerythrin.

Although strong fluorescence makes the R-phycoerythrin (R-PE) proteins increasingly useful in biological and clinical assays, they are subject to nonlinear effects including transitions to collective dark states and photodegradation, which complicate quantitative applications. We report measurements of R-PE fluorescence intensity as a function of incident power, duration of illumination and temperature. Emission intensity in the band at 570 nm is proportional to incident power for low power levels. At higher incident power, the emission at 570 nm is smaller than expected from a linear dependence on power. We propose that R-PE undergoes both reversible emission cessation on a millisecond time scale attributed to transitions to a collective dark state, and irreversible photodegradation on a time scale of minutes. Singlet oxygen scavengers such as dithiothreitol and n-propyl gallate have protective effects against the latter effect but not the former. Electrophoretic analysis of irradiated solutions of R-PE indicates that significant noncovalent aggregation is correlated with photodegradation. A multistate model based on fluorescence measurements and geometric analysis is proposed for the fluorophores in R-PE. The phycobilin fluorophores are partitioned into three groups: the phycourobilins (PUB) absorbing at 490 nm, one group of phycoerythobilins (PEB) absorbing at 530 nm (PEB-530) and another group of PEB absorbing at 560 nm (PEB-560). The two processes that result in the loss of fluorescence intensity are most likely associated with the PEB-560 group.

Fluorescence↗