Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry
适用于CyTOF质谱流式细胞仪的外周血单核细胞（PBMC)细胞因子胞内染色方法
发布: 2015年01月05日第5卷第1期 DOI: 10.21769/BioProtoc.1370 浏览次数: 23226
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
2009
Abstract
In this protocol, we use a CyTOFTM mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium for DNA intercalators. The output data are in the format as .txt and .fcs files, which is compatible with many analysis programs. This protocol could be adapted to include tetramers into the staining panel, but we have not optimized for that purpose.
The principal steps of intracellular cytokine staining are as follows: First, cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the cytokines within the cell. Next, EDTA is added to remove adherent cells from the activation vessel. After washing, antibodies to cell surface markers are added to the cells. The cells are then fixed in paraformaldehyde and permeabilized. We use a gentle detergent, saponin, as the permealization buffer because it is less destructive to surface and intracellular epitopes compared to harsh detergents or methanol. After permeabilization, the metal-conjugated anti-cytokine antibodies are added into the cell suspension. The stained cells are then sequentially introduced into the mass cytometry for signal intensity analysis.
Materials and Reagents
- PBMC (fresh or thawed frozen)
- RPMI-1640 (Hyclone, catalog number: SH30027.01 )
- FBS (Atlanta Biologicals, catalog number: S11150 )
- Pen-strep-Glutamin 100X (Hyclone, catalog number: SV30082.01 )
- Benzonase (2.5 x 105 U/ml) (Pierce, catalog number: 88701 )
- Brefeldin A (Sigma-Aldrich, catalog number: B7651 )
- Monensin (Sigma-Aldrich, catalog number: M5273 )
- 0.5 M EDTA (Hoefer, catalog number: GR-123-100 )
- Sodium azide (10% w/v solution) (Teknova, catalog number: S0209 )
- 16% para-formaldehyde (PFA) (Alfa Aesar, catalog number: 43368 ))
- 10x PBS (ROCKLAND, catalog number: MB-008 )
- BSA (Sigma-Aldrich, catalog number: A7284 )
- Maleimide-DOTA (Macrocyclics, catalog number: B-272 )
- Lanthanum (III) chloride heptahydrate (Sigma-Aldrich, catalog number: 203521 )
- Indium (III) chloride (Sigma-Aldrich, catalog number: 203440 )
- MilliQ water
Note: Beakers or bottles used here are not washed with soap due to barium content of most commercial soaps.
- Phenotyping antibodies (filtered with 0.1 µm spin filters) (Millipore, catalog number: UFC30VV00 )
- Ir-intercalator stock solution (Fluidigm, catalog number: 201192 )
Note: Rh103-intercalator can be used.
- 10x saponin-based permeabilization buffer (eBioscience, catalog number: 00 8333-56 )
- Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
- Ionomycine (Sigma-Aldrich, catalog number: I0634 )
- Phytohemagglutinin (PHA) (Sigma-Aldrich, catalog number: 61764 )
- SEB (Sigma-Aldrich, catalog number: S0812 )
- Anti-CD3/CD28 (various vendors)
- Peptide mixes (JPT)
- Complete RPMI (see Recipes)
- CyPBS (see Recipes)
- CyFACS buffer (see Recipes)
- Live-dead stain (see Recipes)
Equipment
- 96- well round-bottom plates
- 37 °C water bath
- Biosafety cabinet
- Centrifuge
- CO2 incubator at 37 °C
- Calibrated pipettes
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Lin, D., Gupta, S. and Maecker, H. T. (2015). Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry. Bio-protocol 5(1): e1370. DOI: 10.21769/BioProtoc.1370.
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分类
免疫学 > 免疫细胞染色 > 大量细胞计数法
免疫学 > 免疫细胞功能 > 细胞因子
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