मास Cytometry: दैनिक और एक CyTOF मास cytometer पर ट्यूनिंग रनिंग सेल के नमूने के लिए प्रोटोकॉल
Summary
दैनिक और एक CyTOF जन कोशिकामापी के प्रदर्शन की ट्यूनिंग अनुकूलन के लिए आवश्यक कदम वर्णित हैं. इष्टतम नमूना तैयार करने और प्रवाह की दर पर चर्चा कर रहे हैं
Abstract
In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no “spectral overlap” from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.
Protocol
Representative Results
Discussion
पिछले कुछ दशकों के लिए, प्रतिदीप्ति प्रवाह cytometry एकल कक्षों का विश्लेषण करने के लिए एक workhorse विधि किया गया है दोनों सतह अभिव्यक्ति के मामले में और कार्यात्मक assays में. हालांकि, फ्लोरोसेंट रंजक वर्णक्रमीय ओव?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
हम प्रतिक्रिया के लिए डॉ. इवान Newell और डॉ. शॉन Bendall धन्यवाद देना चाहूंगा. हम भी DVS विज्ञान और डॉ. शॉन Bendall मल्टी lanthanide युक्त polystyrene मोती का एक नमूना के लिए धन्यवाद देना चाहूंगा. हम NIH अनुदान 2 AI057229 U19 से धन के लिए आभारी हैं.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| CyTOF mass cytometer | DVS Sciences | ||
| Wash solution | DVS Sciences | 201071 | 0.05% hydrofluoric acid in water |
| Tuning solution | DVS Sciences | 201072 | 0.5 ppm La, Cs, Tb, Tm, Ir, trace nitric acid |
| Eu-containing polystyrene beads | DVS Sciences | 201073 | Contains natural-abundance Eu isotopes; beads supplied at approximately 1 million/ml |
| Multi-lanthanide-containing (La/Pr/Tb/Tm/Lu)- polystyrene beads | DVS Sciences | Not yet commercially-available | Contains natural-abundance isotopes of listed lanthanides |
| Nitric acid (concentrated) | Fisher Scientific | A467-500 | Optima trace-metal pure ICP-MS grade |
| Polystyrene round-bottom tube with cell-strainer cap-5 ml | BD | 352235 | used to filter cell samples before injection |
| Norm-Ject tuberkulin syringe-1 ml | Henke Sass Wolf | 4010-200V0 | silicone-free, latex-free |
| Norm-Ject syringe-3 ml | Henke Sass Wolf | 4010.000V0 | silicone-free, latex-free |
| MilliQ water | 18 MΩ pure water; must not be stored in glass or plastic bottles that have been washed with commercial detergent (due to their high levels of barium present). | ||
| Citranox | Sigma-Aldrich | Z273236 | acid detergent |
| Argon gas | Praxair | AR 5.0UH-T | 99.999% Ultra-high purity |
Riferimenti
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