Explanted कोशिकाओं (ट्रंक तंत्रिका शिखा कोशिकाओं) के प्रवास का विश्लेषण करने के लिए एक दृष्टिकोण वर्णित है. इस विधि सस्ती, कोमल, और प्राथमिक ट्रंक तंत्रिका शिखा सेल संस्कृति के भीतर सेल सेल बातचीत से प्राप्त उन लोगों के रूप में प्रवासी polarity पर दोनों किसी रासायनिक पदार्थ द्वारा किसी जीव की क्रियाशीलता का बढ़ जाना और अन्य प्रभावों से chemotaxis भेद करने में सक्षम है.
Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9.
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions.
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.
ट्रंक एनसीसीएस पर chemotaxis अनुसंधान का आयोजन कारणों की एक श्रृंखला के लिए चुनौती साबित हो गया है. इसलिए, ट्रंक एनसीसीएस NT ट्रंक स्तर की प्राथमिक explantation से प्राप्त किया जाना चाहिए, ट्रंक एनसीसीएस एक विषम स्टेम…
The authors have nothing to disclose.
हम इस पद्धति के विकास के दौरान लिनो किम, स्टीव Guzman और Ujit सत्यार्थी इन बातों के लिए विशेष तकनीकी सहायता के लिए धन्यवाद देते हैं. Myron Hawthorne, रिचर्ड Spengel, और रॉबर्टो Rojas machined यहां इस्तेमाल किया कक्षों और बहुत जरूरी तकनीकी सहायता प्रदान की है. विशेष रूप से, रॉबर्टो Rojas चित्रा 4 का उत्पादन किया. हम भी हैं स्कॉट फ्रेजर अमूल्य ऊपर chemotaxis परख के विकास के लिए पहले सलाह के लिए आभारी हैं. यह काम एक NIH MBRS स्कोर MEdB 5S06GM048680-13 और CSU, Northridge स्नातक थीसिस सहायता कार्यक्रम से एक CW पुरस्कार से आंशिक रूप से समर्थन किया था.
Name of the reagent | Company | Catalogue number | Comments (optional) |
DMEM | Omega Scientific | DM-22 | |
Penicillin Streptomycin Solution | Omega Scientific | PS-20 | 100X Stock Concentration |
L-Glutamine | Omega Scientific | GS-60 | 100X Stock Concentration |
Fetal Bovine Serum | Omega Scientific | FB-11 | Lot# 105247 (or another that is comparable) |
Modified Zigmond chamber | Home made | N/A | Reservoir volume: ~ 160 μl ea; for additional specifications, see Fig. 4 and the supplemental fabrication protocol |
Cell culture dish | Denville | T6040 | 40 x 10 mm |
Fibronectin | BD | 354008 | 10X Stock prepped by diluting 1 mg FN in 1 ml H2O and 9 ml DMEM |
Coverslips | Fisher | 12-548-B | Precleaned; 22 x 22 mm |
L15 medium | Thermo Scientific | SH30525.02 | |
Petroleum Jelly | Comforts | 011110794642 | 100% |
Centrifuge tube | Biologix | 10-9152 | 15 ml |
Dispase | Cell Systems | 4Z0-850 | 10X Stock Concentration |
Syringe | BD | 309602 | 1 ml |
Needle | BD | 305127 | 25 G x 1.5 in. |
Alexa Fluor 488-IgM | Invitrogen | A21042 | Stock is 2 mg/ml; 7 moles dye/mole IgM |
Dissecting Forceps | FST | Misc. | Dumont #5 or 55; straight tipped; stainless steel or titanium |
Tungsten Needle | N/A | N/A | Home made; placed in a pin holder |
Blunt Forceps | Tiemann | 160-18 | Used for transferring embryos to Ringer’s from egg yolk |
Supplemental Protocol: Fabrication of a Modified Zigmond Chamber
Please refer to Figure 4 as a reference for the protocol below: