Table 1.

Comparison of the different technologies for secretion analysis at the single‐cell level

		Spot‐based methods			Cytometry‐based methods		Microfluidic methods
		ELISpot	FluoroSpot	iSCAT microscopy	Flow cytometry	Mass cytometry	Micro‐/ nanowell assays	Microchamber assay	Flow‐based droplet microfluidics	Stationary droplet‐based assay
Applications		B cells T cells Innate immune cells		Cell line	B cells T cells Innate immune cells		B cells T cells Innate immune cells	B cells T cells Innate immune cells	B cells T cells	B cells T cells Macrophages
Readout	Measurement of secretion	Direct	Direct	Direct	Direct or indirect	Direct or indirect	Direct	Direct	Direct	Direct
	Multiplexing [# parameters]	1‐2	1‐4	(Yes) †	3‐15	>40	1‐4	1‐42	1‐4	1‐5
	Quantification	Relative	Relative	Absolute	Relative	Relative	Absolute	Absolute (mostly reported as relative)	Relative	Absolute
	Kinetics	No	No	Yes	No	No	Yes	Yes	No	Yes
Sample size		105 cells/well	105 cells/well	100‐200 cells	105‐107 cells	105‐107 cells	105	104 cells	104‐106 cells	104‐105 cells
Through‐put		Multiple plates	Multiple plates	1 cell	>104 cells/s	102‐103 cells/s	104‐105 cells/array	103 cells/chip	103 cells/s	104‐2 × 105 cells/array
Assay time *		8‐48 h	8‐48 h	1‐3 h	2‐5 h	2‐5 h	2‐5 h	1‐12 h	5‐8 h	1‐3 h
Detection limit	Frequency of secreting cell	∼1/104	1/104	N.A.	1/104	1/104	1/105	1/103	1/104	1/104
	Secreted analyte [molecules]	N.A.	N.A.	1	>40	>400‐500	∼103	∼102	Various, usually >103	>102
Specific cell recovery		No	No	No	No (Yes: FACS)	No	Yes	Yes	Yes	No
Technical complexity [+++, ++, +]		+	+	++	+(+)	+++	+(+)	++(+)	+++	+(+)
Availability [+++, ++, +]		+++	+++	+	+++	+(+)	++	+(+)	++	++
Standardization [+++, ++, +]		+++	+++	+	++	++	++(+)	++(+)	+	++(+)
Costs	Running costs [+++, ++, +]	+	+(+)	+	++	++(+)	+(+)	++	+(+)	+(+)
	Investemen [+++, ++, +]	+	+(+)	++	++(+)	+++	++	++	++	++
References		[19, 20, 22, 43, 140]	[21, 22, 24]	[25, 26]	[46, 69, 72]	[69, 72]	[79, 86, 89, 96]	[5, 12, 106, 115, 117]	[123, 125]	[10, 121]

^†Distribution of molecular weights of secreted proteins can be analyzed

^*Defined as the time from having prepared cells to obtaining raw data

N.A. Not available through measurement

+++ high, ++ medium, + low