Name: preparation of whole bone marrow for mass cytometry analysis of neutrophil-lineage cells
Uploaded: 2019-06-19T14:30:00
Description: Watch this Scientific Journal Video about Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells at JoVE.com

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).

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)
<ol>
	<li>Purchase C57BL/6J mice from a commercial vendor. Feed the standard rodent chow diet and hous...

<ol>
	<li>Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+common+myeloid+progenitor+that+gives+rise+to+all+myeloid+lineages.">A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.</a> Nature. 404, 193-197 (2000).</li><li>Iwasaki, H., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+lineage+commitment+from+the+hematopoietic+stem+cell.">Myeloid lineage commitment from the hematopoietic stem cell.</a> Immunity. 26, 726-740 (2007).</li><li>Manz, M. G., Miyamoto, T., Akashi, K., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+human+clonogenic+common+myeloid+progenitors.">Prospective isolation of human clonogenic common myeloid progenitors.</a> Proceedings of the National Academy of Sciences. 99, 11872-11877 (2002).</li><li>Becher, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-dimensional+analysis+of+the+murine+myeloid+cell+system.">High-dimensional analysis of the murine myeloid cell system.</a> Nature Immunology. 15, 1181-1189 (2014).</li><li>Bendall, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mass+cytometry+of+differential+immune+and+drug+responses+across+a+human+hematopoietic+continuum.">Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.</a> Science. 332, 687-696 (2011).</li><li>Samusik, N., Good, Z., Spitzer, M. H., Davis, K. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+mapping+of+phenotype+space+with+single-cell+data.">Automated mapping of phenotype space with single-cell data.</a> Nature Methods. 13, 493-496 (2016).</li><li>Zhu, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+Early+Unipotent+Neutrophil+Progenitor+with+Pro-tumoral+Activity+in+Mouse+and+Human+Bone+Marrow.">Identification of an Early Unipotent Neutrophil Progenitor with Pro-tumoral Activity in Mouse and Human Bone Marrow.</a> Cell Reports. 24, 2329-2341 (2018).</li><li>Van der Maaten, L. J. P., Hinton, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+High-Dimensional+Data+Using+t-SNE.">Visualizing High-Dimensional Data Using t-SNE.</a> Journal of Machine Learning Research. 9, 2579-2605 (2008).</li><li>Amir, E. A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=viSNE+enables+visualization+of+high+dimensional+single-cell+data+and+reveals+phenotypic+heterogeneity+of+leukemia.">viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.</a> Nature Biotechnology. 31, 545-552 (2013).</li><li>van der Maaten, L., Hinton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+data+using+t-SNE.">Visualizing data using t-SNE.</a> Journal of Machine Learning Research. 9 (85), 2579-2065 (2008).</li><li>Cloos, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Protocol+to+Sample+and+Process+Bone+Marrow+for+Measuring+Measurable+Residual+Disease+and+Leukemic+Stem+Cells+in+Acute+Myeloid+Leukemia.">Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia.</a> Journal of Visualized Experiment. 133, 56386 (2018).</li><li>Bendall, S. C., Nolan, G. P., Roederer, M., Chattopadhyay, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+deep+profiler&#8217;s+guide+to+cytometry.">A deep profiler&#8217;s guide to cytometry.</a> Trends in Immunology. 33, 323-332 (2012).</li><li>Ley, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils:+New+insights+and+open+questions.">Neutrophils: New insights and open questions.</a> Science Immunology. 3 (30), 4579 (2018).</li><li>Ng, L. G., Ostuni, R., Hidalgo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+neutrophils.">Heterogeneity of neutrophils.</a> Nature Reviews in Immunology. , (2019).</li></ol>

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...

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&#8217;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-activated flow cytometry analysis. For example, Ly6G (or Gr-1) and CD11b are considered mature myeloid cell markers and Ly6G+ (or Gr-1+) and CD11b+ cells are routinely removed from BM samples by using magnetic enrichment kits prior to flow cytometry analysis of hematopoietic stem and progenitor cells (HSPCs) or by combining these markers in one dump cocktail channel1,2,3. Another example is that neutrophils are routinely removed from human blood specimen to enrich peripheral blood mononuclear cells (PBMC) for immunological studies. Whole bone marrow isolated from mouse or human, however, is rarely investigated intact for cytometry analysis.
Recently, CyTOF has become a revolutionary tool to investigate the hematopoietic system4,5,6. With CyTOF, the fluorophore-labeled antibodies are replaced by heavy element reporter-labeled antibodies. This method allows for the measurement of over 40 markers simultaneously without the concern of spectrum overlap. It has enabled the analysis of intact biological specimen without pre-depletion steps or a dump channel. Therefore, we can view the hematopoietic system comprehensively with high-content dimensionality from conventional 2-D flow cytometry plots. Cell populations omitted in the past during depletion or gating process can now be brought into light with the high-dimensional data generated by CyTOF4,5. We have designed an antibody panel that simultaneously measures 39 parameters in the hematopoietic system with a focus on the myeloid linage7. Compared to the conventional flow cytometry data, the interpretation and visualization of the unprecedented single-cell high-dimensional data generated by CyTOF is challenging. Computational scientists have developed dimensionality reduction techniques for the visualization of high-dimensional datasets. In this article, we used the algorithm, viSNE, which uses t-Distributed Stochastic Neighbor Embedding (t-SNE) technique to analyze the CyTOF data and to present the high-dimensional result on a 2-dimensional map while conserving the high-dimensional structure of the data8,9,10. On the tSNE plot, similar cells are clustered into subsets and the color is used to highlight the feature of the cells. For example, on Figure 1 the myeloid cells are distributed into several cell subsets based on the similarities of their expression patterns of 33 surface markers resulted from CyTOF (Figure 1)4. Here we investigated mouse bone marrow with our previously reported 39-marker CyTOF panel by viSNE analysis7. viSNE analysis of our CyTOF data revealed an unidentified cell population that showed both HSPC (CD117+) and neutrophil (Ly6G+) characteristics (Figure 2)7.
In conclusion, we present a protocol to process fresh whole bone marrow for CyTOF analysis. In this article, we used mouse bone marrow as an example, while this protocol can also be used to process human bone marrow samples. The details specific to human bone marrow samples are also noted in the protocol as well. The advantage of this protocol is that it contains details such as incubation time and temperature that were optimized to preserve neutrophil-lineage cells in the whole bone marrow to enable investigation on the intact whole bone marrow. This protocol may also be easily modified for fluorescence-activated flow cytometry applications.

<table>
	<tbody>
		<tr>
			<td>CyTOF Antibodies (mouse)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 (Clone 30-F11) -89Y</td>
			<td>Fluidigm</td>
			<td>Cat# 3089005B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human/Mouse CD45R/B220 (Clone RA36B2)-176Yb</td>
			<td>Fluidigm</td>
			<td>Cat# 3176002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD105 (Clone MJ7/18)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 120402; RRID:AB_961070</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD115 (CSF-1R) (Clone AFS98)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 135502; RRID:AB_1937293</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD117/c-kit (Clone 2B8)-166Er</td>
			<td>Fluidigm</td>
			<td>Cat# 3166004B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD11a (Clone M17/4)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 101101; RRID:AB_312774</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11b (Clone M1/70)-148Nd</td>
			<td>Fluidigm</td>
			<td>Cat# 3148003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11c (Clone N418)-142Nd</td>
			<td>Fluidigm</td>
			<td>Cat# 3142003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD127 (IL-7R&#945;) (Clone A7R34)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 133919; RRID:AB_2565433</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD150 (Clone TC1512F12.2)-167Er</td>
			<td>Fluidigm</td>
			<td>Cat# 3167004B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD16.2 (Fc&#947;RIV) (Clone 9E9)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 149502; RRID:AB_2565302</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD162 (Clone 4RA10 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 557787; RRID:AB_647340</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD169 (Siglec-1) (Clone 3D6.112)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 142402; RRID:AB_10916523</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD182 (CXCR2) (Clone SA044G4)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 149302; RRID:AB_2565277</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD183 (Clone CXCR3-173)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 126502; RRID:AB_1027635</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD335 (NKp46) (Clone 29A1.4)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 137625; RRID:AB_2563744</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD34 (Clone MEC14.7)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 119302; RRID:AB_345280</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD41 (Clone MWReg30)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 133919; RRID:AB_2565433</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD43 (Clone S11)-146Nd</td>
			<td>Fluidigm</td>
			<td>Cat# 3146009B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD48 (Clone HM48.1)-156Gd</td>
			<td>Fluidigm</td>
			<td>Cat# 3156012B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD62L (Clone MEL-14)-MaxPar Ready</td>
			<td>ThermoFisher</td>
			<td>Cat# 14-1351-82; RRID:AB_467481</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD71 (Clone RI7217)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 113802; RRID:AB_313563</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD90 (Clone G7)-Purified</td>
			<td>Biolegend</td>
			<td>Cat# 105202; RRID:AB_313169</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse F4/80 (Clone BM8)-159Tb</td>
			<td>Fluidigm</td>
			<td>Cat# 3159009B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Fc&#949;RI&#945; (Clone MAR-1)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 134321; RRID:AB_2563768</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse GM-CSF (MP1-22E9 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 554404; RRID:AB_395370</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse I-A/I-E (Clone M5/114.15.2)-174Yb</td>
			<td>Fluidigm</td>
			<td>Cat# 3174003B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ki67 (Clone B56 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 556003; RRID:AB_396287</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly-6A/E (Sca-1) (Clone D7)-169Tm</td>
			<td>Fluidigm</td>
			<td>Cat# 3169015B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly6B (Clone 7/4)-Purified</td>
			<td>abcam</td>
			<td>Cat# ab53457; RRID:AB_881409</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly-6G (Clone 1A8)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 127637; RRID:AB_2563784</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse NK1.1 (Clone PK136)-165Ho</td>
			<td>Fluidigm</td>
			<td>Cat# 3165018B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Siglec-F (Clone E50-2440 (RUO))-Purified</td>
			<td>BD Biosciences</td>
			<td>Cat# 552125; RRID:AB_394340</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse TCR&#946; (Clone H57-597)-143Nd</td>
			<td>Fluidigm</td>
			<td>(Clone H57-597)-143Nd</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse TER-119/Erythroid Cells (Clone TER-119)-MaxPar Ready</td>
			<td>Biolegend</td>
			<td>Cat# 116241; RRID:AB_2563789</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals, Peptides and Recombinant Proteins </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody Stabilizer</td>
			<td>CANDOR Bioscience</td>
			<td>Cat# 130050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Cisplatin-194Pt</td>
			<td>Fluidigm</td>
			<td>Cat# 201194</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience 1X RBC Lysis Buffer</td>
			<td>ThermoFisher</td>
			<td>Cat# 00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>ThermoFisher</td>
			<td>Cat# 00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>EQ Four Element Calibration Beads</td>
			<td>Fluidigm</td>
			<td>Cat# 201078</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>ThermoFisher</td>
			<td>Cat# AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Omega Scientific</td>
			<td>Cat# FB-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Phosphate Buffered Saline solution</td>
			<td>GE Lifesciences</td>
			<td>Cat#SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercalator-Ir</td>
			<td>Fluidigm</td>
			<td>Cat# 201192B</td>
			<td></td>
		</tr>
		<tr>
			<td>MAXPAR Antibody Labeling Kits</td>
			<td>Fluidigm</td>
			<td>http://www.dvssciences.com/product-catalog-maxpar.php</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# 158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA 1X</td>
			<td>Corning</td>
			<td>Cat# 25-053-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental Model: Organism/Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>Stock No: 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Software Alogrithm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bead-based Normalizer</td>
			<td>Finck et al., 2013</td>
			<td>https://med.virginia.edu/flow-cytometry-facility/wp-content/uploads/sites/170/2015/10/3_Finck-Rachel_CUGM_May2013.pdf</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytobank</td>
			<td>Cytobank</td>
			<td>https://www.cytobank.org/</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofkit v1.r.0</td>
			<td>Chen et al., 2016</td>
			<td>https://bioconductor.org/packages/release/bioc/html/cytofkit.html</td>
			<td></td>
		</tr>
		<tr>
			<td>t-SNE</td>
			<td>van der Maaten and Hinton, 2008</td>
			<td>https://cran.r-project.org/web/packages/Rtsne/index.html</td>
			<td></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

preparation of whole bone marrow for mass cytometry analysis of neutrophil-lineage cells

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.

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...

Watch this Scientific Journal Video about Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells at JoVE.com

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

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.

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 ...

This mass cytometry protocol preserves the integrity of the whole bone marrow, allowing rescue of the information that was lost during conventional sample preparation for analysis of under studied cells. This fast and effortless bone marrow isolation protocol facilitates the acquisition of viable short-lived myeloid cell populations such as rare neutrophil-lineage subsets. Using mass cytometry to identify previously under appreciated immune cells such as neutrophil-lineage cells may have broad implications for a wide variety of diseases.This protocol preserves the integrity of the short lived myeloid cells that are present in the bone marrow and can easily be applied to other tissue types such as the solid tumor. We simplified to protocol to make it as user friendly as possible, but as in all biological studies it may need a few practice runs to manage. When you're doing this the first time, you can get all your reagents ready first and then strictly follow the protocol.If you do experience any issues, you can connect to the CyTOForum and talk to other CyTOF users who can help troubleshoot. For biological sample preparation, simple tricks can make a huge difference with the end result. A visual demonstration is much easier for a new user to grasp the protocol.If you are doing this for the first time, you can get your reagents ready first and then strictly follow the protocol. For bone marrow harvest, place a six to 10 week old C57 black six mouse in the supine position on a sterile surgical pad and use 70%ethanol to sterilize the abdomen and hind limbs. Use a pair of dissecting surgical scissors to open the abdominal cavity and remove the skin to expose the hind limbs.Using a pair of blunt tipped dressing forceps, hold the mouse tibia right below the ankle and use a pair of curved dressing forceps to stabilize the tibia below the blunt tipped dressing forceps. Use the blunt tipped dressing forceps to break the tibia and remove the muscle to expose the bone. Before placing the tibia in cold PBS, move the stabilizing curved dressing forceps to the femur and slide the blunt tipped dressing forceps below the knee joint to hold the kneecap.Gently pull up to dislocate the kneecap and remove the muscle from the kneecap to expose the femur. Hold the exposed femur by the curved dressing forceps and use surgical scissors to cut the femur from the bottom of the bone. Then place the femur in PBS.Next, use an 18 gauge needle to punch a hole in a 0.5 milliliter micro centrifuge tube and place both bones in the tube with the open ends of the bones facing downward into the hole. Place the 0.5 milliliter tube into a 1.7 milliliter micro centrifuge tube and spin the double layered tubes in a micro centrifuge. At the end of the centrifugation, confirm that the bone marrow has been extracted to the bottom of the tube.To stain the bone marrow cells for mass cytometry, re suspend the bone marrow in one milliliter of red blood cell lysis buffer for 10 minutes at room temperature. At the end of the incubation, collect the cells by centrifugation and re suspend the pellet in one milliliter of cold PBS. Filter the cells through a 70 micrometer strainer into a 15 milliliter conical tube and wash the cells with nine milliliters of fresh, cold PBS.Re suspend the bone marrow cell pellet in 10 milliliters of fresh, cold PBS for counting and transfer a five times 10 to the sixth cell aliquot into a new 15 milliliter tube. Collect the cells by centrifugation and re suspend the cells in one milliliter of mass cytometry staining buffer, supplemented with 125 nanomolar cisplatin. After five minutes at room temperature, wash the cells in four milliliters of fresh mass cytometry buffer.Re suspend the pellet in 50 microliters of FC receptor blocking solution. After 10 minutes at four degrees Celsius, add 50 microliters of the antibody cocktail of interest to the cells and gently pipet to mix. The temperature in different incubation steps are very important in preserving the viability of the bone marrow cells.After 30 minutes at four degrees Celsius, wash the cells two times in two milliliters of fresh mass cytometry buffer per wash before re suspending the cells in one milliliter of freshly prepared 1.6%formaldehyde for 15 minutes at room temperature. At the end of the incubation, collect the cells by centrifugation and re suspend the pellet in one milliliter of fixed perm buffer supplemented with 125 nanomolar intercalation solution for an overnight incubation at four degrees Celsius. Before analyzing the cells by mass cytometry, gently vortex and centrifuge the cell suspension before washing the cells in two milliliters of fresh mass cytometry staining buffer.Next, wash the cells two times in one milliliter of distilled water per wash, carefully aspirating the supernatant after the second wash before re suspending the cells at a one times 10 to the sixth cells per milliliter of mass cytometry buffer concentration for mass cytometry analysis. In this T distributed stochastic neighbor embedding plot, the cells across multiple mouse tissues were clustered into subsets based on the similarity of their surface marker expression profiles as measured by a 33 parameter mass cytometry panel. Cells with more similar properties were automatically clustered together based on the expression of these markers on each cell.Using this method, the integrity of the whole bone marrow was preserved, leading to the discovery of a previously unknown cell population that simultaneously co-expresses neutrophil and hematopoietic stem and progenitor cells signature surface markers. This cell population, which was previously omitted in myeloid progenitor studies, due to Ly6G positive cell depletion, exhibits a distinct pattern of surface marker expression. More importantly, these data led to the discovery of a small subset of the Ly6G positive cell cluster that doesn't express Ly6G, but was clustered closely to the CD117 positive Ly6G positive cells, suggesting the similarity of these cells to the neutrophil-lineage based on the expression of the 33 surface markers used in this mass cytometry experiment.A 13 color florescence activated cell sorting panel could then be built, allowing the isolation of the neutrophil progenitors by flow cytometry for downstream functional assays. It is important to perform the bone marrow isolation procedure as quickly as possible and keep the cells at four degrees Celsius during the staining procedure.

preparation-whole-bone-marrow-for-mass-cytometry-analysis-neutrophil

Research

JoVE Journal

Immunology and Infection

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...