common biological elements, enriching for heavy-metal reporter
ions, which are separated by their mass-to-charge ratio in a time-of-
flight mass spectrometer (seeFig. 1b). Based on mass cytometry,
deep phenotyping [40, 41, 56–64], signaling state characterization
[59, 65–68], and cycle analysis [57, 69, 70] at the single-cell level
have been demonstrated.
Although mass cytometry can simultaneously characterize
roughly 100 types of proteins, it still has several drawbacks. In
mass cytometry, cells are atomized and ionized, and thus it remains
infeasible to recover living cells after analysis, and thus it cannot
function as a cell sorter. In addition, due to the dynamics of ion
flight in the mass spectrometer, the throughput of mass cytometry
lags behind that of fluorescence-based instruments. Furthermore,
both fluorescent flow cytometry and mass cytometry cannot pro-
duce absolute copy numbers of intracellular proteins and cannot be
used to estimate secreted proteins at the single-cell level.4 Droplet Cytometry
Since flow cytometry and mass cytometry cannot effectively esti-
mate the secreted proteins from single cells, droplet microfluidics
[71–77] was proposed where aqueous droplets encapsulate individ-
ual cells and confine secreted proteins, which are further character-
ized based on fluorescent detection [78–94](seeFig. 1c). Based on
droplet cytometry, secreted proteins at the single-cell level were
characterized, which includes (1) yellow fluorescent protein mutant
“Venus” of singleE. coli[78]; (2) the activities of enzyme alkaline
phosphatase secreted by singleE. coli[79]; (3) cytokine (IL-10)
secretion of single CD4+CD25+regulatory T cells [80]; (4) intra-
cellular proteins of HRas-mCitrine, expressed within single
HEK-293 cells, and actin-EGFP expressed within single MCF-7
cells [81]; and (5) cytokine (IL-2, IFN-γ, TNF-α) secretion of
single, activated T-cells [82].
Although powerful, droplet cytometry has two limitations,
which need to be further addressed. (1) This technique is notTable 1
(continued)
Technique Key results
Mass cytometry CD4 molecules of fixed whole blood, fixed cryopreserved peripheral blood
mononuclear cells, and lyophilized peripheral blood mononuclear cells were
quantified as 45, 46, and 14 103 per cells [41]
Microengraving The secretion rates of IL-6 of PBMCs were quantified as 6.53.2 molecules/
s (3 h) and 10.67.1 molecules/s (12 h) at the single-cell level [42]
Microengraving The secretion rates off CXCL1, CXCL5, and CXCL8 of single tumor cells were
quantified as 0.3–0.9, 0.6–3.1, and 1.9–4.8 molecules/s, respectively [43]Single-Cell Protein Assays: A Review 299