Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry
采用CyTOF质谱流式细胞仪研究人外周血单核细胞表型
发布: 2015年01月20日第5卷第2期 DOI: 10.21769/BioProtoc.1382 浏览次数: 19571
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2012
Abstract
Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators.
In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOFTM (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.
PBMC (外周血单个核细胞)
Phenotyping (表型)
Mass cytometry (大量细胞计数法)
Materials and Reagents
- Peripheral blood mononuclear cells (PBMC) (fresh, or frozen after Ficoll isolation procedure)
- RPMI (HyClone, catalog number: SH30027.01 )
- FBS (heat-inactivated before use) (Atlanta Biologicals, catalog number: S11150 )
- Pen-Strep-Glutamine (100x) (HyClone, catalog number: SV30082.01 )
- Benzonase (25 x 105 U/ml) (Pierce Antibodies, catalog number: 88701 )
- MilliQ water [no contact with beakers or bottles washed with soap (due to barium content of most commercial soaps)]
- PBS (10x stock) (Rockland, catalog number: MB-008 )
- Bovine serum albumin (BSA) (30% w/v in 0.85% NaCl) (Sigma-Aldrich, catalog number: A7284 )
- 0.5 M EDTA (pH 8.0) (Hoefer, catalog number: GR123-100 )
- Sodium azide (10% w/v solution) (Teknova, catalog number: S0209 )
- 2% para-formaldehyde (PFA) (16% w/v aqueous stock, methanol-free, diluted to 2% in 1x CyPBS) (Alfa Aesar, catalog number: 43368 )
- Maleimide-DOTA (Macrocyclics, catalog number: B-272 )
- 139-Lanthanum (chloride salt) (Sigma-Aldrich, catalog number: 203521 )
- 115-Indium (chloride salt) (Sigma-Aldrich, catalog number: 203440 )
- Phenotyping antibodies (filtered with 0.1 μm spin filters) (MIllipore, catalog number: UFC30VV00 )
- Ir-intercalator stock solution from Fluidigm (catalog number: 201192B ; Rh103-intercalator catalog number: 201103B can be used)
- 10x saponin-based permeabilization buffer (eBiosciences, catalog number: 00-8333-56 )
- Complete RPMI (see Recipes)
- CyPBS (see Recipes)
- CyFACS buffer (see Recipes)
- Live-dead stain (see Recipes)
Equipment
- 37 °C water bath
- 96-well plate: 300 μl well volume (250 μl washes) or 1.2 ml well volume (500 μl washes)
Note: If using a 300 μl plate, do an additional wash step (resuspension and centrifugation) at each point. - Biosafety cabinet
- Centrifuge
- Calibrated pipettes
- ViCell (Beckman Coulter) or Hemocytometer cell counter
- CyTOFTM version 1 mass cytometer (Fluidigm Sciences)
Software
- FlowJo software
- Microsoft Excel
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Leipold, M. D. and Maecker, H. T. (2015). Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry. Bio-protocol 5(2): e1382. DOI: 10.21769/BioProtoc.1382.
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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 > 免疫细胞染色 > 免疫检测
细胞生物学 > 单细胞分析 > 大量细胞计数法
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