Name: preparation of whole bone marrow for mass cytometry analysis of neutrophil-lineage cells
Uploaded: 2019-06-19T14:30:00.000+00:00
Duration: 8 min 9 s
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 house in microisolator cages in a pathogen-free facility.</li>
	<li>Use male mice, 6-10 weeks of age, for experimental purpose. Euthanize by CO2 inhalation followed by the cervical dislocation.</li>
	<li>Place the mouse onto a sterile surgical pad with the abdomen side up. Sterilize the skin of the abdomen and hindlimbs area by spraying 70% ethanol. Use a pair of dissecting surgical scissors to cut the abdominal cavity open.</li>
	<li>Remove the skin to expose hindlimbs. Use a pair of blunt-tip dressing forceps to hold the mouse tibia right below the ankle. Use another pair of curved dressing forceps to stabilize the tibia below the blunt-tip dressing forceps. Break the tibia and expose the bone by ripping off the muscle with the blunt-tip dressing forceps. 
	NOTE: The tibia is loosely attached to the knee joint and can be easily picked out by using the blunt-tip dressing forceps.</li>
	<li>Place the tibia in cold 1x PBS.</li>
	<li>Next, move the stabilizing curved dressing forceps downward to the femur. Slide the blunt-tip dressing forceps below the knee joint and hold the kneecap. Dislocate the kneecap by gently pulling it up. Expose the femur by ripping off the muscle attached to the kneecap. Hold the exposed femur by the curved dressing forceps and cut the femur off from the bottom of the bone using dissecting surgical scissors.</li>
	<li>Place the femur in cold 1x PBS.</li>
	<li>Punch a hole in 0.5 mL microcentrifuge tube with an 18 G needle.</li>
	<li>Place both tibia and femur into the same 0.5 mL microcentrifuge tube with the open end of the bones facing downwards to the hole.</li>
	<li>Place the 0.5 mL tube containing both tibia and femur into a 1.7 mL microcentrifuge tube.</li>
	<li>Spin the double-layered tubes containing both tibia and femur at 5,510 x g for 30 s in the micro-centrifuge.</li>
	<li>Ensure that the BM is extracted from the bones and pelleted at the bottom of the tube. Toss the 0.5 mL tube containing the hallowed bones. The mouse BM is ready for next steps. 
	NOTE: Human BM is harvested at clinical resources as previously described11.</li>
</ol>
2. Stain BM Cells for CyTOF
<ol>
	<li>Resuspend the BM in 1 mL 1x Red Blood Cell (RBC) lysis buffer. For human BM, resuspend the whole BM in a 10x volume of 1x Red Blood Cell (RBC) lysis buffer. Incubate for 10 min at room temperature (RT).</li>
	<li>Spin the tube at 350 x g for 5 min at 4 &#176;C. For human BM, repeat step 2.1 and 2.2 before proceeding to step 2.3.</li>
	<li>Carefully aspirate the supernatant and leave the pellet undisrupted. Resuspend the pellet with 1 mL of cold 1x PBS. Filter the pellet into a 15 mL conical tube through a 70 &#956;m cell strainer. The BM cells are now completely isolated into the tube from the muscle and bone debris. Wash the cells by adding 9 mL of cold 1x PBS into the tube.</li>
	<li>Spin the 15 mL tube at 350 x g for 5min at 4 &#176;C.</li>
	<li>Carefully aspirate the supernatant and resuspend the BM cells with 10 mL cold PBS. Take an aliquot of cells for counting. Count cells using a hemocytometer.</li>
	<li>Aliquot 5 x 106 BM cells into a new 15 mL tube for CyTOF staining.</li>
	<li>Spin the 15 mL tube aliquot at 350 x g for 5 min at 4 &#176;C.</li>
	<li>Carefully aspirate the supernatant and resuspend the BM cells with 125 nM Cisplatin in 1 mL of CyTOF Staining Buffer as a viability indicator for the sample. Incubate for 5 min at RT.</li>
	<li>After incubation, add 4 mL of CyTOF staining buffer to the tube. Spin the tube at 350 x g for 5 min at 4 &#176;C. For human BM, add 10% human AB serum into the CyTOF staining buffer.</li>
	<li>Carefully aspirate the supernatant and resuspend the BM cells with 50 &#181;L of Fc Receptor blocking solution. Incubate for 10 min at 4 &#176;C. Skip this step for human BM.</li>
	<li>Add 50 &#181;L of the homemade CyTOF antibody cocktail5 to the sample so the total staining volume is 100 &#956;L. Gently pipette to mix. Incubate for 30 min at 4 &#176;C. The final volume of the antibody cocktail is 100 &#956;L for both mouse BM and human BM samples.</li>
	<li>Add 2 mL of CyTOF staining buffer to each tube following the incubation to wash the cells, spin the tube at 350 x g for 5 min at 4 &#176;C.</li>
	<li>Repeat step 2.12 for a total of two washes.</li>
	<li>Prepare a fresh 1.6% formaldehyde solution from the 16% stock ampule. Dilute 1 part of the stock formaldehyde with 9 parts of 1x PBS.</li>
	<li>Carefully aspirate the supernatant and resuspend the pellet with 1 mL fresh 1.6% FA solution. Incubate for 15 min at RT.</li>
	<li>Spin the tube at 800 x g for 5 min at 4 &#176;C.</li>
	<li>Carefully aspirate the supernatant and resuspend the cell pellet with 125 nM intercalation solution in 1 mL fix/perm buffer.</li>
	<li>Incubate the sample in intercalation solution overnight at 4 &#176;C.</li>
</ol>
3. Prepare Cells for CyTOF Acquisition
<ol>
	<li>Gently vortex and spin cells at 800 x g for 5 min at 4 &#176;C.</li>
	<li>Wash cells by adding 2 mL of CyTOF staining buffer, spin cells at 800 x g for 5 min at 4 &#176;C and remove the supernatant by aspiration.</li>
	<li>Resuspend cells in 1 mL of diH2O. Reserve a small volume (approximately 10 &#181;L) from each tube to count cells.</li>
	<li>Spin cells at 800 x g for 5 min at 4 &#176;C.</li>
	<li>Repeat step 3.3 and 3.4.</li>
	<li>Carefully aspirate the supernatant and leave the cells in the pellet. The BM cells are now ready for resuspension to the concentration of 1 x 106 cells/mL for CyTOF acquisition.</li>
</ol>

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	<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. 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The authors have nothing to disclose.

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>&lt;strong&gt;CyTOF Antibodies (mouse)&lt;/strong&gt;</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&amp;alpha;) (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&amp;gamma;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&amp;epsilon;RI&amp;alpha; (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&amp;beta; (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>&lt;strong&gt;Chemicals, Peptides and Recombinant Proteins &lt;/strong&gt;</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&amp;nbsp;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>&lt;strong&gt;Experimental Model: Organism/Strains&lt;/strong&gt;</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>&lt;strong&gt;Software Alogrithm&lt;/strong&gt;</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><td></td><td></td><td></td><td></td></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 ...

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Immunology and Infection

9.1K Views.  La Jolla Institute for Immunology. 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.

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

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

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 ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Medicine

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Scalable ATAC-seq Protocol for Chromatin Accessibility in Rodent Neutrophils

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and understanding epigenomic regulation. Neutrophils, as a crucial leukocyte type in immune responses, undergo substantial chromatin architectural changes during differentiation and activation, which significantly impact the gene expression necessary for their functions. ATAC-seq has been instrumental in uncovering key transcription factors in neutrophil maturation, revealing pathogen-specific epigenomic signatures, and identifying therapeutic targets for autoimmune diseases. However, neutrophils' sensitivity to the external milieu complicates high-quality ATAC-seq data production. Here, we propose a scalable protocol for preparing ATAC-seq libraries from rodent bone marrow-derived neutrophils, featuring improved immunomagnetic separation to ensure optimal cell viability and high-quality libraries. The vital elements impacting the library quality and optimization principles for methodological extension are discussed in detail. This protocol will support the researchers who are willing to study the chromatin architecture and epigenomic reprogramming of neutrophils, advancing studies in basic and clinical immunology.

This manuscript outlines a protocol for preparing an ATAC-seq library of neutrophils from murine bone marrow, aiming to guarantee optimal neutrophil viability and high library quality. It offers step-by-step instructions on BMC preparation, immunomagnetic sorting, and library construction, serving as a valuable guide, especially for newcomers to studying neutrophils.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Biology

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

High-Quality Sample Preparation for Multiome 10X Genomics Analysis

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. 
Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. 
The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions.
This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

Author Spotlight: Advancing Biomedical Research Through Single Cell Analysis 

The success of single-cell/single-nuclei transcriptomics and multi-omics largely depends on the quality of cells/nuclei. Therefore, isolating cells/nuclei from tissue and their purification must be highly standardized. This protocol describes the preparation of nuclei from the brain and bone marrow for downstream single-nuclei multiome assay.

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of ...