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.
1. Barcode of Lysed/Fixed Blood Cells
2. Staining of Barcoded Lysed/Fixed Blood Cells and Preparation for Analysis on Mass Cytometry Instrument
Note: Each 1X titer of staining antibody (1 μL of antibody per 100 μL staining reaction), can usually stain 3–4 x 106 cells. Therefore, when all barcoded samples are pooled into one tube, the amount of antibody must be scaled up. If 20 barcoded samples amount to 30 x 106 cells, and each 1X titer can stain 3–4 x 106 cells, the barcoded sample only requires a 10X titer, as opposed to staining each sample individually, which would require a 20X amount of antibody (1X per individual tube). The concentration of the antibody to cell number should be carefully titrated for each individual antibody cocktail (not discussed here).
The authors have nothing to disclose.
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. |