यहाँ हम संवर्धन एक glial फीडर परत की उपस्थिति में चूहे cortical न्यूरॉन्स के लिए एक प्रोटोकॉल प्रदान करते हैं. सभ्य न्यूरॉन्स स्थापित polarity और synapses बनाने, और इलेक्ट्रोफिजियोलॉजी, इमेजिंग कैल्शियम, सेल अस्तित्व assays, immunocytochemistry, और आरएनए / / प्रोटीन अलगाव डीएनए जैसे विभिन्न अनुप्रयोगों में प्रयोग के लिए glia से अलग किया जा सकता है.
This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20×106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.
इस प्रोटोकॉल glia कोशिकाओं की उपस्थिति में संवर्धन चूहे प्राथमिक cortical न्यूरॉन्स के लिए एक तरीका प्रदान करता है, जबकि न्यूरॉन्स आसानी से प्रयोगात्मक विश्लेषण के लिए पृथक की अनुमति. एक स्वस्थ neuronal phenotype के glia स?…
The authors have nothing to disclose.
लेखकों पिछले प्रयोगशाला के सदस्यों को जो इस प्रोटोकॉल का शोधन और पिछले कुछ वर्षों में समर्थन के लिए NIH (DA19808 और DA15014 ओम) योगदान धन्यवाद. अन्ना 1 Abt "अंतःविषय और translational neuroAIDS में अनुसंधान और प्रशिक्षण" (T32 MH078795) के एक साथी है, इस प्रकार, इस काम के हिस्से में रुथ एल Kirschstein राष्ट्रीय अनुसंधान सेवा 5T32MH079785 पुरस्कार के तहत राष्ट्रीय स्वास्थ्य संस्थान के द्वारा समर्थित किया गया था.
Reagent | Concentration |
---|---|
Glucose | 16 mM |
Sucrose | 22 mM |
NaCl | 135 mM |
KCl | 5 mM |
Na2HPO4 | 1 mM |
KH2PO4 | 0.22 mM |
HEPES | 10 mM |
pH | 7.4 |
Osmolarity | 310±10 mOsm |
Table 1. Dissection Medium.
Reagent | Concentration |
---|---|
DMEM | 90% |
FBS | 10% |
Gentamicin | 50 μg/mL |
Tabe 2. Glia Plating Medium.
Reagent | Concentration |
---|---|
DMEM | 90% |
Horse Serum | 10% |
Table 3. Neuron Plating Medium.
Reagent | Concentration |
---|---|
DMEM | 98% |
N2 Supplement | 1% |
1M HEPES | 1% |
Ovalbumin | 50 mg/100mL |
Table 4. N2 Medium.
Reagent | Concentration |
---|---|
Boric Acid | 50 mM |
Sodium Borate | 12.5 mM |
Table 5. Borate Buffer (for poly-lysine).
Reagent | Company | Catalogue number |
---|---|---|
High glucose Dulbecco’s Modified Eagle Medium (DMEM) |
Invitrogen | 11995-073 |
Fetal Bovine Serum (FBS), Heat-inactivated | Hyclone | 26400-044 |
Horse Serum, Heat-inactivated | Hyclone | H1138 |
Gentamicin (50mg/mL) | Invitrogen | 15750-060 |
N2 Supplement (100x) | Invitrogen | 17502-048 |
HEPES buffer solution | Invitrogen | 15630-080 |
Albumin from chicken egg white, Grade VI (Ovalbumin) |
Sigma-Aldrich | A2512 |
2.5% Trypsin | Invitrogen | 15090-046 |
0.5% Trypsin-EDTA (10X) | Invitrogen | 15400-054 |
Deoxyribonuclease I from bovine pancreas (DNase) |
Sigma-Aldrich | D-5025 |
Paraplast | Fisher | 12-646-106 |
Poly-L-lysine | Sigma-Aldrich | P1274 |
Cytosine-β-D-arabinofuranoside hydrochloride | Sigma-Aldrich | C6645 |
Stereomicroscope | Leica | Leica ZOOM 2000 |
Cover glasses, Circles, 15 mm, Thickness 0.13-0.17 mm |
Carolina | 633031 |
Thermanox sheets | Grace BioLabs | HS4550 |
Large forceps | Biomedical Ricerca Instruments |
70-4000 |
Fine-tipped No.5 forceps | Fine Science Tools |
91150-20 |
Pattern No.1 forceps | Biomedical Ricerca |
10-1400 |
Instruments | ||
Scissors, straight, sharp-blunt | Biomedical Ricerca Instruments |
28-1435 |
Micro Dissecting scissors | Biomedical Ricerca Instruments |
11-2070 |
Micro Dissecting Curved scissors | Biomedical Ricerca Instruments |
11-1395 |