Estimating transfection efficiency in differentiated and undifferentiated neural cells

Abstract

Objective: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before.

Results: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.

Keywords: Lipofection; Neuroblastoma cell lines; Primary cortical astrocytes; Primary cortical neurons; Transfection efficiency.

Fig. 1

Transfection efficiency in B35 and B104 neuroblastoma cell lines. Representative images of transfected undifferentiated neuroblastoma cell lines. a (GFP⁺) B35 cells. b (GFP⁺) B104 cells. c Quantitative analysis of the percentage of GFP⁺ cells among the total DAPI⁺ cells (total population). For B35, transfection efficiency at 24 hpt was 12.06% SEM ± 0.97 and 10.67% SEM ± 1.34 at 48 hpt. Similar rates were achieved for B104 cells, (mean value 12.2% SEM ± 0.80) at 24 hpt and (mean value 11.5% SEM ± 1.24) at 48 hpt. Cells were transfected at 60–80% confluency in 6-well plates for the indicated periods and about 9–11 fields were imaged per well. Each experiment was done in replicates (2 or 3 wells/condition) from 3 independent cultures. Between (8000 and 24,000 cells) were quantified per experiment. Data represents mean value ± SEM. Nuclear staining DAPI (blue). GFP (green)

Fig. 2

Transfection efficiency of astrocytes in primary cortical cultures. a Representative images of transfected astrocytes (GFP⁺/GFAP⁺) in AE and in AC cultures. AE cultures transfected at 60–80% confluency in 6-well plates for the indicated periods and about 10 fields were imaged per well. AC cultures transfected at DIV2 for 48 h in 24-well plates and 7 fields were imaged per well. Each experiment was done in replicates (2 or 3 wells/condition). Total number of cells quantified in each experiment was; (3500–7800 cells) for AC and (2900–7800 cells) for AE. b Quantitative analysis of the percentage of GFP⁺/GFAP⁺ cells among the total DAPI⁺ cells (total population, blue). Mean transfection efficiency of astrocytes in AE was obtained from 3 and 2 independent cultures for 24 hpt and 48 hpt experiments, respectively. Mean transfection efficiency of astrocytes in AC was obtained from 2 independent cultures. Data represents mean value ± SEM

Fig. 3

Transfection efficiency of neurons in primary cortical cultures. Representative images of transfected neurons (GFP⁺/MAP2⁺) in AC (a) and in AF (b) cultures. Cortical cultures were transfected at DIV7/8 in 24-well plates for the indicated periods. c Quantitative analysis of the percentage of GFP⁺/MAP2⁺ cells among the total DAPI⁺ cells (total population, blue). The transfection rate for AC was 1.3% ± 0.3 at 24 hpt and 2.6% ± 0.4 at 48 hpt. About 6–9 fields were imaged per well and each experiment was done in triplicates (3 wells/condition). Mean transfection efficiency of neurons in AC was obtained from 7 and 3 independent cultures for 24 hpt and 48 hpt experiments, respectively. About (5164–20,014 cells) were quantified in each experiment for AC. As for the AF transfection rate was 4.9% ± 1.5 at 24 hpt and 6.4% ± 1.9 at 48 hpt. Mean transfection efficiency of neurons in AF was obtained from 5 and 3 independent cultures for 24 hpt and 48 hpt experiments, respectively. About (476–6097 cells) were quantified in each experiment for AF. Data represents mean value ± S.E.M