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

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][3][4][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. Video LinkThe video component of this article can be found at http://www.jove.com/video/4398/ ProtocolAll cell samples for the CyTOF must be fixed and permeabilized. This enables greater entry of the iridium-containing DNA intercalator, and also prevents cell lysis during the MilliQ water wash and resuspension steps immediately before injecting into the mass cytometer.

Barcoding of Live Human Peripheral Blood Mononuclear Cells for Multiplexed Mass Cytometry

“…The CyTOF ® instrument (version 1, Fluidigm) was started, tuned, and cleaned as described before on a daily basis (8), using tuning and wash solution (Fluidigm). Ultrapure argon was supplied by Praxair (Danbury, CT).…”

“…For mass cytometry, variability in the machine performance (1) as well as in the sample preparation and staining procedure can be partially overcome by daily tuning of the CyTOF ® mass cytometer (8) and by using normalization beads (9). However, standardization of mass cytometry experiments that involve the comparison of multiple samples or stimulation conditions, should ideally based on exactly identical conditions for sample preparation and acquisition.…”

Algorithmic Tools for Mining High-Dimensional Cytometry Data

“…Stringent barcoding schemes and carefully monitoring and optimizing the concentration of the injected cell sample can eliminate the majority of doublets and help alleviate this problem (28). Variations in machine performance can also be addressed by standardized daily tuning procedures and metal-loaded beads for normalization of mass cytometry data (29,30). Currently, no computational tool exists for quantifying the quality of data and identifying potentially problematic samples.…”

Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry

“…[14]. The length of the run will be dependent upon the volume of your sample: dilution with MilliQ water to ~1 million cells/mL is recommended for minimizing doublets as well as maximizing sample throughput ( see Note 10).…”