पीत ज्वर मच्छर से Hemocytes एकत्रित और धुंधला के लिए एक प्रोटोकॉल एडीज aegypti</em
Summary
अभी तक सटीक विधि को इकट्ठा करने और वर्णित है मच्छर hemocytes दाग सरलीकृत. हमारे विधि उच्च इंजेक्शन तकनीक की सटीकता एडीज मच्छरों में hemocytes की साफ तैयारी को अलग के साथ छिड़काव की सादगी को जोड़ती है. इस पद्धति का अध्ययन hemocytes के प्रकार के ज्ञान की आवश्यकता होती है और उनके बहुतायत की सुविधा.
Abstract
Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells.
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.
Protocol
Discussion
hemocyte संग्रह विधि यहाँ वर्णित पहले प्रकाशित तरीकों से संशोधित है और एक कम कदम के साथ एडीज मच्छरों से hemocytes की साफ तैयारी को अलग करने के लिए अनुमति देता है. जबकि हमारे विशेष रुचि एडीज मच्छरों में hemocyte आब…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखक जॉन फ्रे और बेन पीटरसन मच्छर पालन के लिए शुक्रिया अदा करना होगा. हम भी क्रिस्टीन डेविस और गैरी Radice hemocyte micrographs साथ मदद के लिए धन्यवाद. इस शोध रिचमंड कला और विज्ञान गर्मियों ए.ए. Qayum और संकाय ए Telang के लिए अनुदान के लिए फैलोशिप विश्वविद्यालय द्वारा वित्त पोषित किया गया था.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Schneider’s Medium | Sigma-Aldrich | S0146 | |
| Fetal bovine serum | Sigma-Aldrich | F0643 | |
| Hema3 stain kit | Fisher | 123-869 | |
| Glass needles (borosilicate with filament) |
Sutter Instrument | BF100-78-10 | 1.0mm O.D. and 0.78mm I.D. |
| Needle puller | Sutter Instruments | Model P-87 | |
| MicroInjector | Tritech Research | MINJ-PD | |
| Needle holder | Tritech Research | MINJ-4 |
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