A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

Published: February 29, 2024

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 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. 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 th…

Divulgazioni

The authors have nothing to disclose.

Materials

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.

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Citazione di questo articolo
A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells. J. Vis. Exp. (Pending Publication), e21988, doi: (2024).

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