Intraperitoneal संक्रमण के बाद इम्यून कोशिकाओं तस्करी प्रतिदीप्त prion छड़ घंटे निगरानी
Summary
यहाँ हम तुरंत शुद्ध द्वारा intraperitoneal टीका के बाद और fluorescently संक्रमित मस्तिष्क तो उनके और इंजेक्शन साइट से तेज आंदोलन की निगरानी और इन घटनाओं mediating कोशिकाओं निस्र्पक सामग्री से एकत्रित prion छड़ लेबलिंग prion और प्रतिरक्षा कोशिकाओं द्वारा तेज तस्करी की निगरानी के लिए एक उपन्यास परख का वर्णन.
Abstract
Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.
Protocol
Discussion
यहाँ हम vivo में है कि बहुत परिधीय prion संक्रमण में जल्दी घटनाओं की निगरानी की सुविधा में टैगिंग और ट्रैकिंग prions के लिए एक प्रोटोकॉल का प्रदर्शन. इस प्रोटोकॉल बहुत पूर्व लेबलिंग अत्यधिक समृद्ध prion inoculum द…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
हम स्टीव McBryant और जेफ Hansen ultracentrifugation और पट्टी Kiser के साथ माउस से निपटने के साथ मदद के लिए मदद के लिए धन्यवाद. स्नायविक रोग और स्ट्रोक के राष्ट्रीय स्वास्थ्य संस्थान के राष्ट्रीय संस्थान, 5R01NS056379 02-अनुदान इस काम वित्त पोषित.
Materials
| Material Name | Typ | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| CWD-infected elk brain | Private elk farm in Colorado | Use any non-human prion-infected brain | ||
| Blender | Oster | 6694-015 | Use any commercial blender | |
| Centrifuge | Sorvall | SS34 rotor | Use any centrifuge /rotor that can reach 3000 x g and hold ≥ 500 ml volumes | |
| Ultracentrifuge | Beckman | 50.2 Ti rotor | Use any ultracentrifuge /rotor that can reach 100,000 x g and hold ≥ 500 ml volumes | |
| Bradford Reagent | Sigma-Aldrich | B6916 | ||
| Complete mini protease inhibitor cocktail | Roche | 11 836 170 001 | ||
| Sonicator | Misonix | MP4000X | Use any horn or probe sonicator set to ~70% max power | |
| DyLight antibody Labeling kit | Thermo Scientific | 53050 | ||
| microcentrifuge | Eppendorf | 55430R | Use any refrigerated microcentrifuge that can achieve 13,000x g | |
| centrifugal filter columns | Millipore | Microcon YM-100 | Use any filter or dialysis membrane with 100 Kd molecular weight cutoff | |
| 8-40 week-old FVB mice | Charles River | 207 | Use any inbred mouse strain | |
| 1 μm red fluorescent beads | Phosphorex | 2307 | Use any fluorescent bead ≤ 10 μm | |
| RPMI 1640 medium | Invitrogen | 11875-093 | ||
| 40 μm cell strainer | Falcon | 352340 | ||
| fluorescent antibodies | BD pharmingen | Various | Use any fluorescent antibody appropriate for your application. | |
| flow cytometer | Dakocytomation | CyanADP | Use any flow cytometer capable of multicolor fluorescence detection |
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