The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.
Prior to hippocampal isolation
Before beginning the hippocampal isolation, make sure that all the tools are sterile. Spray down the culture hood with 70% ethanol, and place the tools inside the hood. You will need 6- and 10-cm Petri dishes, sterile poly-L coated glass coverslips, pipettors and tips, disposable pipettes, and an electric pipettor. From this point on, remember to use correct sterile technique.
Hippocampal Isolation
Hippocampal cell dissociation
Fura-2 Calcium Imaging of Hippocampal Neurons
This protocol was developed as a modification of the seminal work of Gary Banker and Kim Goslin 1. This book is an essential resource for anyone interested in culturing cells — not only the neuron-enriched cultures described in the current protocol.
The three most critical factors in cell culturing are: sterility, speed, the choice of medium used.
Sterility – a laminar flow/sterile hood, aseptic conditions, and a sterile incubator are required. Contamination at any step is detrimental not only to the current experiment you are working on, but also the subsequent work that may have been planned. After an incubator becomes contaminated, it may take weeks (or a month) to get it cleaned and sterile. Contamination, while not immediately evident at a visible level, will definitely affect cell viability and cell physiology. Sterility is imperative.
Speed – the health of cultured cells is strongly tied to the amount of time the cells are not in an atmosphere and medium controlled environment. Therefore, it is essential that the amount of time required to remove cells from the animal until the cells are in the plating medium is at a minimum. Because numerous steps cannot be altered (time in incubating in trypsin or being washed), what can be altered is the amount of time required to remove the cells from the brain. Practice the dissection often. The amount of time required for this step needs to be reproducible.
Choice of medium – there are numerous proprietary mediums, one of which is Neurobasal. Prior to the days of using Neurobasal, glial conditioned medium (which needed to be made separately – this is discussed in the book by Banker and Goslin 1) was required. Regardless, it is important that you know what is in the medium that is being used. Along with Neurobasal, a supplement such as B-27 is often used. Make sure that the constituents of these medium and supplements are known, as they may affect the properties of the cells. It is standard practice in my lab to use serum free/charcoal stripped/phenol red free medium to avoid steroid hormones (given that a large portion of the work performed in my lab investigates the actions of steroid hormones). This concept — know what is in the solutions you are using — is critical for all steps in cell culturing.
JLN was supported by MH 68347.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Antibiotic antimitotic | Invitrogen | 15240062 | ||
B-27 Supplement | Invitrogen | 17504044 | Serum free | |
Boric Acid | Sigma | B6768 | ||
Dnase I | Sigma | DN25 | ||
Fura-2, AM cell permeant | Molecular Probes | F1221 | ||
Glucose | Sigma | G7528 | ||
HBSS (10X) | Invitrogen | 14185052 | ||
HEPES | Invitrogen | 15630080 | ||
L-Glutamine | Sigma | G7513 | ||
MEM | Invitrogen | 51200038 | ||
Neurobasal | Invitrogen | 12348017 | Without phenol red | |
Poly-L-Lysine Hydrobromide | Sigma | P6282 | ||
Pyruvic Acid | Sigma | P2256 | ||
Sodium Pyruvate | Sigma | P2256 | ||
Sodium Tetraborate | Sigma | |||
Trypsin, 2.5% (10X) | Invitrogen | 15090046 |