A method to evaluate the efficiency of transfection reagents in an adherent zebrafish cell line

Abstract

We present a simple and robust method to evaluate the transfection efficiency of commercially available transfection reagents intended to be established for use in nonmammalian cell lines. To illustrate the method, we compare the ability of four different reagents to transfect the embryonic zebrafish cell line Z3. Z3 cells were seeded in a 96-well plate and simultaneously transfected in several variations by using minimum volumes of transfection reagent and a vector DNA encoding an amplified version of green fluorescent protein (GFP). After 24 and 48 h, transfection efficiency was determined by a dual fluorescence plate reader measurement of GFP and Hoechst 33342 fluorescence, an indicator of cell density. Of the four different reagents tested, certain variations of JetPrime(™) reagent and X-tremeGene(™) HP reagent produced the highest fluorescence signal per cell after 24- and 48-h incubation, respectively. The simultaneous multivariate setup enables comparing different reagent/DNA combinations at different time points well, independent of cell growth variability or seeding density.

Keywords: Hoechst 33342; Z3; maxGFP; transfection; zebrafish cell culture.

FIG. 1.

Example for a multivariate optimization layout. Schematic layout of different transfection variations (see Table 1) used for optimizing three transfection methods in one 96-well plate simultaneously. Variations of factor 1 are represented as wells filled with light (variation A) and dark (variation B) gray color. In the case of Lipofectamine LTX, the amount of DNA (μg) and PLUS reagent (μL) varied with X-tremeGene HP, the volume of transfection mix added to the wells varied; and with JetPrime the volume of reagent (μL) used varied. Variations of factor 2 are organized in well columns. At 24 and 48 h post transfection (hpt) incubation time points are organized in double rows. White wells held nontransfected cells used as controls for the cell viability assay. The crossed-out wells represent empty wells used for holding the Hoechst staining blank. The layout for the Matra-A transfection was similarly organized on an additional 96-well plate (layout not shown).

FIG. 2.

Calibration curve of Hoechst 33342-saturated zebrafish Z3 cells seeded at various cell densities. Hoechst fluorescence was recorded after 80-min incubation with the Hoechst 33342 nuclear stain (1 μg/mL) in Z3 cells which were seeded at various densities in triplicates 24 h earlier. The curve fitted dashed line has a correlation factor R²=0.994. Inset: The incubation time until Hoechst fluorescence signal becomes saturated. Fluorescence was recorded every 10 min after incubation started in triplicate wells holding 5×10⁴ zebrafish Z3 cells/cm². The dotted line marks the 80-min incubation time point. All values represent blank-corrected means±SEM of three independent cell culture preparations.

FIG. 3.

Quantified transfection. Bar charts show maxGFP fluorescence normalized to cell density at 24 h (A) and 48 h (B) post transfection. For each of the four transfection reagents factors 1 and 2 were varied according to Table 1. Significant differences (p≤0.05) between variations within each transfection reagent are discerned by letters with or without a prime symbol. Values represent means±SEM of five independent cell culture preparations.

FIG. 4.

Cell viability 48 h post transfection. Bar charts represent cell densities of different transfection variations translated from Hoechst measurements 48 h post transfection and displayed as a percentage of nontransfected control cell density (dashed line). Cell viability was not significantly altered after 24 h post transfection (data not shown). Significant differences (p≤0.05) between variations within each transfection reagent are discerned by letters with or without a prime symbol. Values represent means±SEM of five independent cell culture preparations.

FIG. 5.

Hoechst 33342–stained nuclei and maxGFP expression recorded after 24 h in Z3 cells. Representative images of Z3 cells 24 h after transfection (18.4% efficiency) show fluorescence signals of Hoechst 33342 (A), maxGFP expression (B), and an overlay of both (C). Scale bar: 100 μm.