PHOSPHO FLOW CYTOMETRY: SINGLE-CELL SIGNALING NETWORKS IN NEXT-GENERATION DRUG DISCOVERY AND PATIENT STRATIFICATION - Flow Cytometry in Drug Discovery and Development - page 318

of the individual atoms that made up the cell, including the elemental tags used to
label the antibody.
. The resulting elemental ions are transferred into a high-vacuum region, neutral
species are removed, and the cloud becomes focused into a beam that is
transmitted further into the mass analysis region.
. The beam of elemental ions then passes through a radio frequency-only
quadrupole mass filter that removes the highly abundant, low molecular
weightelementalions(i.e.,C,N,O,S,Na,K,Cl,andCa)thatcomposethe
bulk of biomass. This is a necessary step, as these elements would overwhelm
the detector hindering the analysis of the low-abundance heavy metal isotope
tags.
. The heavier elemental ions (approximately 100 Da and higher) are then trans-
mitted into the time-of-flight (TOF) mass analysis region where the beam is
sampled and mass measured (scanned) approximately 80,000 times per second.
The very fast TOF scan rate is the component of the platform that allows single-
cell measurement on the order of 100-1000 times per second.
. The average ion cloud corresponding to a single cell event has a duration of
25-30 scans. Moreover, this ion cloud has a signal intensity distribution that is
mainly Gaussian. Using these principles, the single-cell signal is extracted from
the raw data by integrating the signal from each parameter measured, reporting it
as a single value for each cellular event measured. This is quite similar to the
protocols in modern fluorescent cytometric analyzers.
. The data are saved as text and in FCS3.0 formats, the latter being compatiblewith
conventional flow cytometry data processing software.

On the CyTOF instrument, the TOF mass analyzer is optimized to measure
elements approximately between 100 and 250 Da. There is currently a panel of 30
nonbiological, rare elemental metal isotopes commercially available for use as
reporters in mass cytometry analysis. The metal isotopes are listed in Table 15.1.
The majority of these metals belong to the lanthanide series of elements. These metal

TABLE 15.1 CommonMetal Isotopes Used inCompensation-Free
Mass Cytometry Analysis
Isotope/Elemental Symbol

139 La 150 Nd 160 Gd 170 Er

141 Pr 151 Eu 164 Dy 171 Yb

142 Nd 152 Sm 165 Ho 172 Yb

144 Nd 153 Eu 166 Er 174 Yb

145 Nd 154 Sm 167 Er 175 Lu

146 Nd 156 Gd 168 Er 176 Yb

147 Sm 158 Gd 169 Tm 197 Au

148 Nd 159 Tb