A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

Abstract

Source: Baxter, R. M., et al. Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry. J. Vis. Exp. (2018).

This video highlights a single-cell proteomic method that employs heavy-metal-conjugated antibodies for distinct cell barcoding. Subsequently, immunostaining and mass cytometry are applied to evaluate immune phenotypic and functional alterations in human whole blood-derived immune cells.

Protocol

1. Barcode of Lysed/Fixed Blood Cells

1. Thaw the lysed/fixed cell samples from the -80 °C storage slowly on ice; a maximum of 20 samples can be labeled with unique barcodes and pooled using this system. Dilute the 10X barcoding perm buffer by 1:10 with PBS; make enough buffer for ~3 mL per sample.
2. Fill one non-sterile trough with the CSM and one with the 1X barcoding perm buffer. Add 1 mL of ice-cold CSM to freshly thawed samples, mix thoroughly with a pipette, and transfer to the respective pre-labeled polypropylene cluster tubes.
3. Take a 10 μL sample to count cells using an automated cell counter or hemocytomet....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ruxolitinib	Santa Cruz	SC-364729A	Stock Conc: 10 mM; Final Conc: 5 μM
R848	Invivogen	tlrl-r848-5	Stock Conc: 1 μg/μL; Final Conc: 1 μg/mL
LPS-EK	Invivogen	tlrl-eklps	Stock Conc: 1 μg/μL; Final Conc: 0.1 μg/mL
Sterile PBS	Lonza	17-516F
Lyse/Fix Buffer	BD biosciences	558049	Stock Conc: 5X; Final Conc: 1X (dilute in ddH2O)
BD Phosflow perm/wash buffer I	BD biosciences	557885	Stock Conc: 10X; Final Conc: 1:10 (dilute in ddH2O)
RPMI	Gibco	21870-076
Sodium Azide (NaN3)	Sigma-Aldrich	S-8032	Stock Conc:>99.9%; Final Conc: 0.0002
Protein Transport Inhibitor (PTI)	eBiosciences	00-4980-93	Stock Conc: 500X; Final Conc: 1X
DNA Intercalator	Fluidigm	201192B	Stock Conc: 500 μM; Final Conc: 0.1 μM
MaxPar Barcode Perm Buffer	Fluidigm	201057	Stock Conc: 10X; Final Conc: 1X
20-plex Pd Barcode Set	Fluidigm	S0014	Stock Conc: n/a; Final Conc: n/a
Antibodies used for Mass Cytometry
Surface markers
CD1c	Biolegend	L161	Mass: 161
CD3	BD	UCHT1	Mass: 144
CD4	Biolegend	SK3	Mass: 174
CD7	BD	M-T701	Mass: 149
CD8	Biolegend	SK1	Mass: 142
CD11b	Fluidigm	ICRF44	Mass: 209
CD11c	BD	B-ly6	Mass: 152
CD15	BD	HI98	Mass: 115
CD14	Biolegend	M5E2	Mass: 154
CD16	eBioscience/Thermo	B73.1	Mass: 165
CD19	Santa Cruz	SJ25C1	Mass: 163
CD21	Biolegend	Bu32	Mass: 141
CD27	BD	L128	Mass: 155
CD38	Fluidigm	HIT2	Mass: 172
CD45 total	Biolegend	HI30	Mass: 89
CD45RA	Biolegend	HI100	Mass: 153
CD56	Miltenyi	REA196	Mass: 168
CD66	BD	B1.1/CD66	Mass: 113
CD86	Fluidigm	IT2.2	Mass: 150
CD123	Fluidigm	6H6	Mass: 143
CD278/ICOS	Biolegend	C398.4A	Mass: 156
CD179/PD1	Biolegend	EH12.2H7	Mass: 162
IgD	Biolegend	IA6-2	Mass: 146
IgM	Biolegend	MHM-88	Mass: 151
CXCR5	BD	RF8B2	Mass: 173
HLADR	Biolegend	L243	Mass: 167
Cytokines
IL-1α	Biolegend	364-3B3-14	Mass: 147
IL-1β	Biolegend	H1b-98	Mass: 169
IL-1RA	Santa Cruz	AS17	Mass: 157
IL-6	Biolegend	MQ2-13A5	Mass: 164
IL-8	BD	E8N1	Mass: 160
IL-12/IL-23p40	Biolegend	C8.6	Mass: 171
IL-17A	Biolegend	BL168	Mass: 148
IL23p19	eBioscience/Thermo	23dcdp	Mass: 176
MIP1β	BD	D21-1351	Mass: 158
MCP1	BD	5D3-F7	Mass: 170
IFNα	Miltenyi	LT27:295	Mass: 175
IFNγ	Biolegend	4S.B3	Mass: 165
PTEN	BD	A2B1	Mass: 159
TNFα	Biolegend	Mab11	Mass: 166
Note: If the manufacturer is stated as Fluidigm, this antibody was purchased from Fluidigm with metal pre-conjugated. If the manufacturer is stated as other than Fluidigm, this antibody was self-conjugated using the MaxPar Multi-Metal Labeling Kit (Fluidigm Cat: 201300) according to manufacturer protocol.