Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.
Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.
Primary neurons are one of the hardest cell types to transfect as they are postmitotic and are very sensitive to micro-environmental changes. There are four commonly used types of methods for expression of exogenous genes and short-hairpin RNAs (shRNAs) in these cells1. Each has its own advantages and disadvantages. For example, electroporation is usually performed on freshly isolated neurons2, as cells must be transferred into cuvettes for transfection. Virus infection can usually achieve very high efficiency3, but is more labor-intensive and risky for operators. Many lipid-mediated transfection reagents are available commercially, with varying degrees of success in neurons and different levels of cytotoxicity.
Calcium phosphate transfection represents a convenient and economical method for introducing foreign genes into neurons. The method was first used to introduce adenovirus DNA into mammalian cells by Graham and Van Der Eb (1973)4. Transfection was performed by mixing calcium chloride with recombinant DNA in a phosphate buffer. This allows the formation of DNA/calcium phosphate precipitates which, when gradually dropped onto a monolayer of cells, adhere to the cell surface, are taken up by endocytosis and finally enter the nucleus5. This process would lead to the expression of introduced foreign genes in the target cell. Typical efficiencies of calcium phosphate transfection range between 0.5-5%6-8. However, with careful optimization and consistent execution of the experimental protocol, it is possible to reach a transfection efficiency of almost 50%. Here we describe our detailed protocol for calcium phosphate transfection of primary hippocampal neurons, which are cocultured with astroglial cells in a sandwich format9.
There are several key parameters that need to be carefully controlled for consistently successful transfections10,11. The most critical parameter for calcium phosphate transfection is the pH value of 2x HBS, which in our hands usually varies between 7.10-7.15. We recommend making three batches of stocks with pH values in 0.05 increments to account for the difference between pH meters. Alternatively, the Clontech mammalian transfection kit provides 2x HBS that consistently yields good efficiency. Keep in m…
The authors have nothing to disclose.
This work is supported by NIH grant NS065183 and start-up funds from the Rutgers Robert Wood Johnson Medical School.
Dumont #5 forceps | Fine Science Tools | 11251-20 | |
Fine scissors | Fine Science Tools | 14090-09 | straight / sharp 8.5 cm |
Vannas-Tubinger spring scissors | Fine Science Tools | 15008-08 | angled / sharp / 8.5 cm / 5 mm cutting edge |
Standard pattern forceps | Fine Science Tools | 11000-16 | |
Student surgical scissors | Fine Science Tools | 91401-14 | blunt / 14.5 cm |
Spring scissors | Fine Science Tools | 15006-09 | angled to side / 9 cm / 10 mm cutting edge |
Isoflurane | Webster Veterinary | 14043-225-06 | |
Poly-L-lysine | Sigma | P2636 | |
MEM | Sigma | M2279 | |
100 mM Sodium pyruvate | Sigma | S8636 | |
Glucose | Sigma | G8270 | |
10x HBSS | Sigma | H4385 | |
1 M HEPES, pH 7.3 | Gibco/Invitrogen | 15630-080 | |
GlutaMAX | Gibco/Invitrogen | 35050-061 | |
Neurobasal Media | Gibco/Invitrogen | 21130-049 | |
B27 Supplement (50x) | Gibco/Invitrogen | 17504-044 | |
N2 Supplement | Gibco/Invitrogen | 17502-048 | |
Ovalbumin | Sigma | A5503 | |
Penicillin/Streptomycin | Gibco/Invitrogen | 15070-063 | |
2.5% Trypsin | Gibco/Invitrogen | 15090-046 | |
DNase I | Sigma | DN-25 | |
Cytosine arabinoside | Calbiochem/EMD Millipore | 251010 | |
Kynurenic acid | Sigma | K3375 |