बैक्टीरियल विष प्रेरित लाइव सेल प्रतिदीप्ति माइक्रोस्कोपी का उपयोग प्रतिक्रियाएँ की विज़ुअलाइज़ेशन

Summary

रीकॉम्बीनैंट से कोलेस्ट्रॉल बाध्यकारी विष streptolysin हे सफ़ाई के लिए तरीके<em> ई. कोलाई</em> और यूकेरियोटिक कोशिकाओं जीने बंधन विष के दृश्य का वर्णन कर रहे हैं. विष के स्थानीयकृत वितरण को लक्षित कोशिकाओं में तेजी से और जटिल परिवर्तन के विष जीव विज्ञान के उपन्यास पहलुओं का खुलासा करने को प्रेरित करता है.

Abstract

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type¹. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines². Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.

Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism^3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation⁴. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines^5,6,7. A link between IL-1β release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes⁸. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Protocol

1. Streptolysin हे के शुद्धीकरण (SLO) BL21 सोने की 0.5 एल लेग शोरबा में प्लाज्मिड pBADgIII – SLOhis 9 युक्त कोशिकाओं के 20 मिलीलीटर रातोंरात संस्कृति टीका लगाना और 500 μl 50 मिलीग्राम / एमएल एम्पीसिलीन जोड़ें. 225 rpm पर 37 डिग्री सेल्स…

Representative Results

10 आमतौर पर 7 -10 8 यू / मिलीलीटर SLO 4 मिलीग्राम / एमएल के एक प्रोटीन एकाग्रता के साथ प्राप्त किया जा सकता है. विष की सेल के लिए आवश्यक राशि कोशिका प्रकार से भिन्न होता है, लेकिन आम तौर पर है यू 125-500 मिलीग्रा?…

Discussion

तकनीक यहाँ वर्णित जीवाणु विषाक्त पदार्थों को प्रतिरक्षा कोशिकाओं की प्रतिक्रियाओं की परीक्षा के लिए अनुमति देते हैं. सबसे महत्वपूर्ण कदम से निपटने और विष के dosing है. विष गतिविधि अत्यंत चर उसी तैयारी के…

Materials

Name of the reagent	Company	Catalogue number	Comments (optional)
Ni-NTA agarose	Qiagen	30210
polymixin-agarose	Sigma	P1411-5ML
Zeba Desalt Spin col	Fisher	PI-89891
sheep RBCs	Fisher	50-415-688
pBADgIII-SLO	N/A	N/A	see ref9
Cy5 monoreactive dye	GE Healthcare	PA25001
Fura2-AM	Life Technologies	F1221
Calcein AM/ Ethidium homodimer	Life Technologies	L3224
Anti-CD11c-APC	BD Biosciences	550261
collagen-coated glass-bottom dish	Mattek	P35GCol-1.5-10-C
femto-tip II	Fisher	E5242957000
Microloader	Fisher	E5242956003
dextran Alexa 555	Life Technologies	D34679
Injectman NI 2	Eppendorf	920000029
FemtoJet	Eppendorf	5247 000.013
			Table 1. List and source of specific reagents and equipment needed. Specific equipment and reagents used in this protocol, along with company and catalogue number are listed.
			Buffer Composition Step Used Lyse/Wash 50 mM NaH2PO4 300 mM NaCl 10 mM imidazole, pH 8.0 1.4 Tris/salt 50 mM Tris, pH 8.0 300 mM NaCl 1.7 Elution buffer 50 mM Tris, pH 8.0 300 mM NaCl 250 mM imidazole 1.8 RBC Assay buffer 0.3% BSA 2 mM CaCl2 10 mM HEPES, pH 7.4 2.1 buffer R/B RPMI cell culture medium 2 mM CaCl2 0.5% BSA 3.1
			Table 2. List of buffers used in this protocol. The buffers used, their composition and the first step at which they are used in the protocol are listed.