ثنائي الصفائح المشارك ثقافة الجرذ الخلايا العصبية الابتدائية القشرية والدبقية
Summary
هنا نقدم مشروع بروتوكول لزراعة الخلايا العصبية الفئران القشرية في وجود طبقة المغذية الدبقية. الخلايا العصبية مثقف إقامة قطبية وخلق نقاط الاشتباك العصبي ، ويمكن فصلها عن الدبقية لاستخدامها في مختلف التطبيقات ، مثل الكهربية ، والتصوير الكالسيوم والمقايسات بقاء الخلية ، وكيمياء سيتولوجية مناعية ، وRNA / DNA / عزل بروتين.
Abstract
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.
Protocol
Discussion
يوفر هذا البروتوكول طريقة لزراعة الخلايا العصبية الفئران القشرية الأولية في وجود خلايا الدبقية ، بينما يسمح لتكون الخلايا العصبية معزولة بسهولة لأغراض التحليل التجريبي. لدعم التنمية دبق النمط الظاهري العصبية a صحية ، في حين أن الاستجابات العصبية تحوير أيضا لعلاجات…
Disclosures
The authors have nothing to disclose.
Acknowledgements
الكتاب أشكر أعضاء المختبر السابقين الذين ساهموا في صقل هذا البروتوكول والمعاهد الوطنية للصحة للحصول على الدعم على مر السنين (DA19808 وDA15014 إلى OM). آنا أبت 1 هو زميل في "تدريب والبحوث متعددة التخصصات في neuroAIDS بالحركة" (T32 – MH078795) ، وبالتالي تم دعم هذا العمل في جزء من المعاهد الوطنية للصحة تحت L. روث الجائزة الوطنية كيرشتاين خدمة أبحاث 5T32MH079785.
Materials
| 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 Research Instruments |
70-4000 |
| Fine-tipped No.5 forceps | Fine Science Tools |
91150-20 |
| Pattern No.1 forceps | Biomedical Research |
10-1400 |
| Instruments | ||
| Scissors, straight, sharp-blunt | Biomedical Research Instruments |
28-1435 |
| Micro Dissecting scissors | Biomedical Research Instruments |
11-2070 |
| Micro Dissecting Curved scissors | Biomedical Research Instruments |
11-1395 |
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