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

摘要: Background Enhanced green fluorescent protein (EGFP) is used extensively to assess gene expression on cells; however, quantification of this by flow cytometry has been limited the unavailability calibration standards. Thus, we characterized response an experimental set EGFP standards environmental changes and then quantitate EGFP, in molecules equivalent soluble fluorochrome (MESF) units, a transfected Molt-4 T cell line cytometry. Methods Characterization standards: were equilibrated suspension solutions having pH range 5.0–9.0, temperatures 37–80°C, osmolalities 100–600 mOsm/kg. Quantification cells: For transfections, cells incubated with two different concentrations (0.2 μg 0.4 μg) pEGFP-N2 vector was quantified after 48 h using cytofluor technique standard curve known solutions. Results The fluorescence intensity increased from 5.0 9.0 remained relatively constant 37°C 65°C, 100 600 After transfection, populations high low averaged 8,098 ± 584 MESF 3,808 375 respectively. No significant differences observed comparing values obtained Cytofluor (high: 8,791 492 MESF; low: 4,082 398 MESF). Conclusions Our data demonstrate feasibility quantify cells. Our results emphasize importance monitoring effects both samples when quantifying living © 2004 Wiley-Liss, Inc.