Virus vectors have become very useful tools for transgene delivery. Viruses have found numerous applications in the biomedical sciences, including developmental neuroscience. This protocol describes the infection of primary neuronal cultures with virus vectors.
STEP 1
Before virus infection, aspirate half of the previous medium for changing the media, and add the lentivirus according to the MOI=5-20 or add the AAV virus according to the MOI=10
4-10
5, then mix gently and place it in the incubator.
STEP 2
Gently aspirate the culture medium after 8-12 hours of lentivirus infection，please note that avoid sucking up completely which will cause the neurons damage due to air oxidation. Quickly add the previous preheated culture medium.
STEP 3
After cultivating for 2-3 days, determine the infection efficiency of the lentivirus infection on the target cells under a fluorescent microscope. Generally speaking, the infection experiment was successful when more than 80% of the neurons can be visible.
STEP 4
After 3 days, neurons can be used for follow-up work according to the experimental design, such as western blot, qPCR, immunohistochemistry and electrophysiological experiments.
Figure 1. Gene knockdown experiment on cultured hippocampal neurons with lentivirus infection (Dittgen et al. 10.1073/pnas.0407976101)
Figure 2. Cultured cortical neurons infected with different subtypes of AAV virus（Virology. 2008 March 1; 372(1): 24–34. doi:10.1016/j.virol.2007.10.007）
Note: 1) Before virus infection, it is necessary to observe the neuron morphology to ensure that the neuron is in good condition, and then infect with the virus.
2) The condition of primary neuron is very important, poor condition of the lentivirus will cause more neuron death. In addition, because primary cultured neurons are relatively sensitive, it is not recommended to add polybrene to enhance infection whether it is AAV or lentiviral infection particles.
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