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

Abstract

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°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 g and 0.4 g) 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

Quantitative flow cytometry of ZAP‐70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome

“…We chose to employ absolute MESF units, which allowed numerical quantitation of the signal when compared with that of calibration beads, rather that to that of background. Such an approach for ZAP-70 analysis could offer a highly accurate and robust assay that could be standardized across multiple laboratories depending on uniformity of instrument settings and performing strictly defined fixing and staining protocols (19,20,28,29). Here, we present a novel quantitative flow cytometry analysis that enables electronic calculation of the average amount of ZAP-70 The analysis done according to the conventional method (11) shows that CLL patients with 20% ZAP-70 have a shorter time to progression than those with <20% positive patients (P ¼ 0.0026).…”

“…MESF has been used to assess the number of TdT, CD10, and CD19 molecules per normal B-cell precursors and in B-CLL, with a major difference in the level of expression of TdT, CD10, and CD19 between normal bone marrow and B-lineage ALL blasts (32) and to monitor changes in the amount of CD5, CD19, CD20, CD23, and HLA DR in treated CLL patients when compared with untreated patients at diagnosis (33). MESF has also been used to quantitate expression of T-cell antigens in normal peripheral blood lymphocytes (34) and EGFP, CD4, CD8, and CD45 in Molt T cells (28). Calibration of the ZAP-70 assay can now be easily performed, as standards and calibration beads are readily available.…”

Development and optimization of an EGFP-based reporter for measuring the general stress response inListeria monocytogenes

“…EGFP folding and chromophore formation is also faster 22 and its structure was reported to be stable over osmolarity and temperature ranges. 23 One of the key factors in considering a successful reporter of s B activity in L. monocytogenes is the choice of an effective s B -dependent promoter. In the pioneering in vitro studies of sporulation in B. subtilis Haldenwang and Losick (1979) 24 used the ctc gene in their transcription assays, which led to the discovery of one of the first bacterial alternative sigma factor, s B .…”

Reevaluation of quantitative flow cytometric analysis for TLR2 on monocytes using F(ab′)2 fragments of monoclonal antibodies

“…Currently, they are divided into two methodologies. One is the method of using a set of bead particles that are coupled with a particular amount of fluorochromes, which allows the conversion of relative MFI fluorescence values to units of molecules of equivalent soluble fluorochrome per cell (30)(31)(32). Another one is using a set of particles that are coupled with mouse or other animal's IgG to mimic the antibody-labeled cells.…”