This Application Note describes how the Agilent 2100 bioanalyzer and the cell fluorescence LabChip® kit can be used to determine the efficiency of transfection of mammalian cells using green fluorescent protein (GFP) as a reporter molecule by on-chip staining. Transfection of CHO-K1 cells with an expression vector encoding GFP was performed and cells were stained on-chip with the live-dye CBNF (carboxynaphthofluorescein). The histogram quality and the percent transfected cells determined are in good agreement with data obtained using a conventional flow cytometer. Detailed protocols and reagent recommendations for analyzing transfection reactions are provided. Speed of the on-chip staining procedure, high reproducibility of the chip results, low cell consumption and ease-of-use are advantages the Agilent 2100 bioanalyzer offers for monitoring transfection efficiency using GFP.
This document details a method to detect green fluorescent protein (GFP) expression in transfected mammalian cells using the Agilent 2100 bioanalyzer and the cell fluorescence LabChip kit. The protocol involves on-chip staining with the live-dye CBNF to distinguish live cells from dead ones, allowing for the accurate determination of transfection efficiency.
The text provides a detailed experimental protocol for analyzing CHO-K1 cells transfected with a GFP expression vector. It compares the results obtained from the bioanalyzer with those from conventional flow cytometry and fluorescence microscopy, highlighting the speed, reproducibility, and low cell consumption advantages of the on-chip staining procedure.
Approximately 600 to 800 cell events are counted for each sample.
The final concentration of CBNF in the sample well is 10 uM.
The chip must be incubated for 15 minutes at room temperature in a humidified chamber.