Article

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer.

Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University.
Journal of Visualized Experiments 01/2012; DOI: 10.3791/4398
Source: PubMed

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.

1 Follower
 · 
159 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well. Copyright © 2015 by The American Association of Immunologists, Inc.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background Human immunodeficiency virus (HIV), which causes acquired immune deficiency syndrome (AIDS), has killed many people in the world. But sub-Saharan African is the hardest-hit and it is a home to 71% of the world's HIV infected people. However, very little is known about HIV cases the Republic of South Sudan. The review examines the prevalence of HIV infection, transmission and treatment and government intervention initiatives to mitigate the spread of the disease in South Sudan. Methods We searched the database in www.ncbi.nlm.nih.gov/pubmed for articles on HIV using the terms human immunodeficiency virus, acquired immune deficiency syndrome and Republic of South Sudan. We searched the government documents and website of the ministry of health for articles containing HIV and AIDS. Results HIV prevalence in South Sudan ranges between 3.7 and 15.5%. There is however, a generalized heterogeneity in its prevalence, with towns along the border registering higher cases. Major towns within the country also experience higher HIV cases. With increasing cases of self-stigmatization, denial and discrimination of those living with HIV, many more people are expected to acquire and transmit the disease. Conclusion The rate of HIV infection is on the increase in the Republic of South Sudan. This has been attributed to dismal poverty, illiteracy, lack of awareness programs, having multiple sexual partners and widow inheritance.
    01/2013; 12(3):55–62. DOI:10.1016/j.hivar.2013.07.002