Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.
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Method Article
Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells
In This Article
Summary
Abstract
In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.
Introduction
In the past few decades, cytometry methods have been a powerful tool to investigate the hematopoietic system in the BM. These methods include fluorescence-activated flow cytometry and the new method of CyTOF using heavy metal-labeled antibodies. They have led to discoveries of many cell types in a heterogeneous biological specimen by identification of their unique surface marker expression profiles. Increased spectrum overlaps that’s associated with more channels leads to higher data inaccuracy in fluorescence-activated flow cytometry applications. Therefore, unwanted cells are routinely removed in order to enrich cell populations of interest for fluorescence-ac....
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Protocol
All experiments followed approved guidelines of the La Jolla Institute for Allergy and Immunology Animal Care and Use Committee, and approval for the use of rodents was obtained from the La Jolla Institute for Allergy and Immunology according to criteria outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health.
1. Harvest Mouse Bone Marrow (BM)
- Purchase C57BL/6J mice from a commercial vendor. Feed the standard rodent chow diet and house in microisolator cages in a pathogen-free facility.
- Use male mice, 6-10 weeks of age, for experimental purpose. Euthanize by CO2
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Results
Figure 1 is presented as an example result from CyTOF experiments. On this tSNE plot the cells across multiple mouse tissues were clustered into subsets based on the similarity of their surface marker expression profiles measured by a 33-parameter CyTOF panel. Cells with more similar properties were automatically clustered together such as the neutrophils, macrophages, or the DCs based on the expression of the 33 markers on each cell.
Figure 2.......
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Discussion
In past decades, fluorescence-based flow cytometry was used as the main method to study cellular lineages and heterogeneity1,2,3. Although flow cytometry has provided multi-dimensional data, this method is limited by choices of parameters and spectral overlap. To overcome the weakness of flow cytometry we took advantage of CyTOF, which uses heavy metal isotopes instead of fluorophores to label antibodies that eliminates crosstal.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank the LJI Flow Cytometry core for assistance with mass cytometry procedure. This work was supported by NIH grants R01HL134236, P01HL136275, and R01CA202987 (all to C.C.H) and ADA7-12-MN-31 (04) (to C.C.H. and Y.P.Z).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| CyTOF Antibodies (mouse) | |||
| Anti-Mouse CD45 (Clone 30-F11) -89Y | Fluidigm | Cat# 3089005B | |
| Anti-Human/Mouse CD45R/B220 (Clone RA36B2)-176Yb | Fluidigm | Cat# 3176002B | |
| Anti-mouse CD105 (Clone MJ7/18)-Purified | Biolegend | Cat# 120402; RRID:AB_961070 | |
| Anti-mouse CD115 (CSF-1R) (Clone AFS98)-Purified | Biolegend | Cat# 135502; RRID:AB_1937293 | |
| Anti-Mouse CD117/c-kit (Clone 2B8)-166Er | Fluidigm | Cat# 3166004B | |
| Anti-mouse CD11a (Clone M17/4)-Purified | Biolegend | Cat# 101101; RRID:AB_312774 | |
| Anti-Mouse CD11b (Clone M1/70)-148Nd | Fluidigm | Cat# 3148003B | |
| Anti-Mouse CD11c (Clone N418)-142Nd | Fluidigm | Cat# 3142003B | |
| Anti-mouse CD127 (IL-7Rα) (Clone A7R34)-MaxPar Ready | Biolegend | Cat# 133919; RRID:AB_2565433 | |
| Anti-Mouse CD150 (Clone TC1512F12.2)-167Er | Fluidigm | Cat# 3167004B | |
| Anti-mouse CD16.2 (FcγRIV) (Clone 9E9)-Purified | Biolegend | Cat# 149502; RRID:AB_2565302 | |
| Anti-Mouse CD162 (Clone 4RA10 (RUO))-Purified | BD Biosciences | Cat# 557787; RRID:AB_647340 | |
| Anti-mouse CD169 (Siglec-1) (Clone 3D6.112)-Purified | Biolegend | Cat# 142402; RRID:AB_10916523 | |
| Anti-mouse CD182 (CXCR2) (Clone SA044G4)-Purified | Biolegend | Cat# 149302; RRID:AB_2565277 | |
| Anti-mouse CD183 (Clone CXCR3-173)-Purified | Biolegend | Cat# 126502; RRID:AB_1027635 | |
| Anti-mouse CD335 (NKp46) (Clone 29A1.4)-MaxPar Ready | Biolegend | Cat# 137625; RRID:AB_2563744 | |
| Anti-mouse CD34 (Clone MEC14.7)-Purified | Biolegend | Cat# 119302; RRID:AB_345280 | |
| Anti-mouse CD41 (Clone MWReg30)-MaxPar Ready | Biolegend | Cat# 133919; RRID:AB_2565433 | |
| Anti-Mouse CD43 (Clone S11)-146Nd | Fluidigm | Cat# 3146009B | |
| Anti-Mouse CD48 (Clone HM48.1)-156Gd | Fluidigm | Cat# 3156012B | |
| Anti-mouse CD62L (Clone MEL-14)-MaxPar Ready | ThermoFisher | Cat# 14-1351-82; RRID:AB_467481 | |
| Anti-mouse CD71 (Clone RI7217)-Purified | Biolegend | Cat# 113802; RRID:AB_313563 | |
| Anti-mouse CD90 (Clone G7)-Purified | Biolegend | Cat# 105202; RRID:AB_313169 | |
| Anti-Mouse F4/80 (Clone BM8)-159Tb | Fluidigm | Cat# 3159009B | |
| Anti-mouse FcεRIα (Clone MAR-1)-MaxPar Ready | Biolegend | Cat# 134321; RRID:AB_2563768 | |
| Anti-mouse GM-CSF (MP1-22E9 (RUO))-Purified | BD Biosciences | Cat# 554404; RRID:AB_395370 | |
| Anti-Mouse I-A/I-E (Clone M5/114.15.2)-174Yb | Fluidigm | Cat# 3174003B | |
| Anti-Mouse Ki67 (Clone B56 (RUO))-Purified | BD Biosciences | Cat# 556003; RRID:AB_396287 | |
| Anti-Mouse Ly-6A/E (Sca-1) (Clone D7)-169Tm | Fluidigm | Cat# 3169015B | |
| Anti-Mouse Ly6B (Clone 7/4)-Purified | abcam | Cat# ab53457; RRID:AB_881409 | |
| Anti-mouse Ly-6G (Clone 1A8)-MaxPar Ready | Biolegend | Cat# 127637; RRID:AB_2563784 | |
| Anti-Mouse NK1.1 (Clone PK136)-165Ho | Fluidigm | Cat# 3165018B | |
| Anti-Mouse Siglec-F (Clone E50-2440 (RUO))-Purified | BD Biosciences | Cat# 552125; RRID:AB_394340 | |
| Anti-Mouse TCRβ (Clone H57-597)-143Nd | Fluidigm | (Clone H57-597)-143Nd | |
| Anti-mouse TER-119/Erythroid Cells (Clone TER-119)-MaxPar Ready | Biolegend | Cat# 116241; RRID:AB_2563789 | |
| Chemicals, Peptides and Recombinant Proteins | |||
| Antibody Stabilizer | CANDOR Bioscience | Cat# 130050 | |
| Bovine Serum Albumin | Sigma-Aldrich | Cat# A4503 | |
| Cisplatin-194Pt | Fluidigm | Cat# 201194 | |
| eBioscience 1x RBC Lysis Buffer | ThermoFisher | Cat# 00-4333-57 | |
| eBioscience Foxp3 / Transcription Factor Staining Buffer Set | ThermoFisher | Cat# 00-4333-57 | |
| EQ Four Element Calibration Beads | Fluidigm | Cat# 201078 | |
| Ethylenediaminetetraacetic acid (EDTA) | ThermoFisher | Cat# AM9260G | |
| Fetal Bovine Serum | Omega Scientific | Cat# FB-02 | |
| HyClone Phosphate Buffered Saline solution | GE Lifesciences | Cat#SH30256.01 | |
| Intercalator-Ir | Fluidigm | Cat# 201192B | |
| MAXPAR Antibody Labeling Kits | Fluidigm | http://www.dvssciences.com/product-catalog-maxpar.php | |
| Paraformaldehyde | Sigma-Aldrich | Cat# 158127 | |
| Sodium azide | Sigma-Aldrich | Cat# S2002 | |
| Triton X-100 | Sigma-Aldrich | Cat# X100 | |
| Trypsin EDTA 1x | Corning | Cat# 25-053-Cl | |
| Experimental Model: Organism/Strains | |||
| Mouse: C57BL/6J | The Jackson Laboratory | Stock No: 000664 | |
| Software Alogrithm | |||
| Bead-based Normalizer | Finck et al., 2013 | https://med.virginia.edu/flow-cytometry-facility/wp-content/uploads/sites/170/2015/10/3_Finck-Rachel_CUGM_May2013.pdf | |
| Cytobank | Cytobank | https://www.cytobank.org/ | |
| Cytofkit v1.r.0 | Chen et al., 2016 | https://bioconductor.org/packages/release/bioc/html/cytofkit.html | |
| t-SNE | van der Maaten and Hinton, 2008 | https://cran.r-project.org/web/packages/Rtsne/index.html | |
References
- Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 404, 193-197 (2000).
- Iwasaki, H., Akashi, K. Myeloid lineage commitment from the hematopoietic stem cell. Immunity. 26....
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