Materials and methods
Preparation of primary cortical cultures and neuroblastoma cell lines
Animal handling and culling was carried out in accordance with schedule 1 of the standard guidelines on human killing of animals, whereby pregnant dams were euthanized by a trained personnel using CO2, while fetuses were euthanized by decapitation with surgical scissors. Cortical neurons were cultured from embryonic day 17.5 or 18.5 (E17.5/18.5) CD1 or C57BL/6 mouse pups as previously described [12]. Dissociated cortices were plated at a density of 13 × 104 cells/cm2 in a 24-well plate. Three types of cortical cultures were generated: astrocyte-containing (AC), astrocyte-free (AF) and astrocytes enriched cultures (AE). Undifferentiated rat B35 and B104 neuroblastoma cells were routinely grown in 25 cm2 flasks and subcultured when confluent. Detailed information is provided in Additional file 1.
Transient transfection and plasmids
Primary cortical cultures (on DIV7/8) or neuroblastoma cells (B35 and B104) were transiently transfected using Lipofectamine 2000 (Invitrogen) and pEGFP-N3 (Clenotech) following a previously described protocol [11]. Primary cortical cells were transferred to serum free non-trophic medium 2–4 h prior to transfection. A DNA (µg): Lipofectamine (µl) ratio of (1:3.88) was applied to each well. For AE cultures and neuroblastoma cell lines, the same protocol was applied to cells at a confluency of 60–80% with two modifications: (1) cells were seeded in 6-well plates, (2) the recommended transfection medium was replaced with Opti-MEM™ I Reduced Serum Medium (Gibco, France). Neuroblastoma cell lines and AE cultures were transfected with pEGFP-N3:Lipofectamine ratio of (3 µg:11.65 µl/well). Whereas, AC and AF cultures were grown in 24-well plates and transfected with pEGFP-N3:Lipofectamine ratio of (0.6 µg:2.33 µl/well). At the end of the transfection period, the medium on the cells was replaced with either cell-conditioned media (primary cortical cultures) or fresh serum-free growth medium (for neuroblastoma and AE cultures) to prevent cytotoxicity. Finally, cells were fixed either 24 or 48 h post-transfection (hpt), before staining with the desired anti-bodies.
Cell viability assessment
Cell viability was determined using FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, for Flow Cytometry (Invitrogen) according to the manufacture’s protocol. A more detailed description of the protocol is provided in Additional file 1.
Immunocytochemistry and imaging
Immunofluorescence was performed as previously described [12]. All anti-bodies used in this study are listed in Additional file 1: Table S1. Non-saturated images were acquired using Nikon epi-fluorescent microscope under a 10× objective lens. Image analysis was performed with (Image j) program. For primary cortical cultures phenotyping, we first quantified the total number of cells in each well, by staining the cells with the nuclear marker (DAPI) and excluding ones with pyknotic nuclei. Then percentage of neurons or astrocytes was determined by calculating the percentage of NeuN+ and GFAP+ cells in the total population (DAPI+).
Transfection efficiency in the dividing cells studied here (B35/B104), comprising of a homogenous population of cells, was determined by quantifying the percentage of GFP+ cells within the total (DAPI+) population. Whereas in primary cortical cultures, the efficiency was determined at a cell-type level using cell identity markers.
Results and discussion
Determining viability of transfected cells
Different gene delivery strategies can have various cytotoxic effects which in turn can confound determination of transfection efficiency. We therefore have assessed toxicity of Lipofectamine-mediated transfection in our preparations. In general low cytotoxicity was observed for all tested neural cultures except AE. Lipofectamine showed the lowest toxicity in neuroblastoma B35 and B104 with cell death increase of only 5.2 and 7.1% from the baseline, respectively (Additional file 1: Fig. S1A, B). Similarly, good survival rates were achieved for AC and AF with only 11 and 12% increase in cytotoxicity levels over baseline, respectively (Additional file 1: Fig. S1C, D). As for AE, relatively high toxicity was observed even in non-transfected cells despite of their normal morphological appearance (Additional file 1: Fig. S2). The overestimation of dead cells may be due to false positive events resulting from PI staining of RNA (see Additional file 1). Considering the above, we applied an alternative method for estimating cytotoxicity in AE using DAPI nuclear staining and measured death by calculating the percentage of pyknotic nuclei in the total population. A modest toxicity was observed with a 17% increase in cell death levels over baseline (Additional file 1: Fig. S1E).
Determining transfection efficiency in neuroblastoma cells
Undifferentiated rodent neuroblastoma cell lines such as B35 and B104, are useful in vitro models of CNS for studying important aspects of neurobiology associated with neurodevelopmental processes such as differentiation, neurite outgrowth, cell migration and cell death [13, 14].
Efficiencies were determined at 24 and 48 hpt to allow for high transgene expression [15] (Fig. 1a, b). In B35 cells, efficiencies were comparable for both time points. Moreover, similar rates were achieved for B104 cells (Fig. 1c).
Phenotyping of mouse primary cortical cultures
Unlike cell-lines, primary cultures contain heterogeneous population of cells. Therefore, we carried out cell phenotyping at DIV7/8 using the typical neuronal and astrocytic markers (NeuN and GFAP) (Additional file 1: Fig. S3A). The proportion of neurons and astrocytes was calculated as the percentage of NeuN+ or GFAP+ cells/total cell population (DAPI+ nuclei). Dead cells with pyknotic nuclei were excluded.
Our AF cultures were almost completely devoid of astrocytes consisting of 99% neurons, while the AC cultures were composed of 90% neurons and approximately 10% astrocytes. The AE cultures, on the other hand, were predominantly astrocytic containing about 95% GFAP+ cells (Additional file 1: Fig. S3B). These findings are consistent with previous studies [16, 17].
Determining transfection efficiency in primary astrocytes
The ability to generate different types of cortical cultures with various (neurons vs glia) proportions facilitates the study of these cells in the context of pure or mixed population cultures. This is important when investigating cell-type specific responses or cell–cell interaction.
Here we have assessed the transfection efficiency in astrocytes when cultured alone (AE cultures) (Fig. 2a). Both tested time points gave comparable transfection efficiencies (Fig. 2b).
To evaluate the transfection efficiency of astrocytes in our AC cultures, we performed transfections on DIV2. Introducing the transfection mixture at this developmental stage have been observed to preferentially transfect astrocytes in this type of cell culture [11]. Assessment of the transfection efficiency 48 hpt, revealed that the GFP+ cells were predominantly astrocytes (74%) (Fig. 2a and Additional file 1: Fig. S4A). The overall percentage of transfected cells (total GFP+/NeuN+ and GFP+/GFAP+) was 6.4% of which 1.6% were neurons and 4.7% were astrocytes (Fig. 2b).
Determining transfection efficiency in primary neurons
We decided to assess the transfection efficiency of neurons at DIV7/8, a developmental stage typically chosen for carrying out transfections in primary neurons [8,9,10, 12, 18, 19].
The protocol applied here reportedly selects neurons over astrocytes [11, 17], and in our AC cultures it almost exclusively transfects neurons (only 3 GFP+/GFAP+ cells were detected vs 1330 GFP+ neurons in a total of 84,685 cells analyzed) (Fig. 3a and Additional file 1: Fig. S4B). The achieved transfection rate was 1.3% and 2.6% at 24 and 48 hpt, respectively (Fig. 3c).
As for the AF (cultures), a higher transfection rate was obtained (4.9% at 24 hpt and 6.4% at 48 hpt) (Fig. 3b, c). To our knowledge, transfection rate has not been previously determined in rodent primary AF “pure neuronal” cultures. However, the achieved transfection efficiencies in our primary neuronal cultures are within the previously reported range (1–5%) using the same method [2, 11, 17].
In an attempt to improve transfection efficiency, we trialed various Lipofectamine:DNA ratios and tested transfecting cells at DIV4 instead of DIV7/8. However, none of the tested conditions gave better results (see Additional file 1).