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

Summary

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.

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-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 channel^1,2,3. 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 system^4,5,6. 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 CyTOF^4,5. 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 data^8,9,10. 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.

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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)

1. Purchase C57BL/6J mice from a commercial vendor. Feed the standard rodent chow diet and house in microisolator cages in a pathogen-free facility.
2. Use male mice, 6-10 weeks of age, for experimental purpose. Euthanize by CO₂ inhalation followed by the cervical dislocation.
3. 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.
4. 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.
5. Place the tibia in cold 1x PBS.
6. 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.
7. Place the femur in cold 1x PBS.
8. Punch a hole in 0.5 mL microcentrifuge tube with an 18 G needle.
9. 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.
10. Place the 0.5 mL tube containing both tibia and femur into a 1.7 mL microcentrifuge tube.
11. Spin the double-layered tubes containing both tibia and femur at 5,510 x g for 30 s in the micro-centrifuge.
12. 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.

2. Stain BM Cells for CyTOF

1. 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).
2. Spin the tube at 350 x g for 5 min at 4 °C. For human BM, repeat step 2.1 and 2.2 before proceeding to step 2.3.
3. 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 μm 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.
4. Spin the 15 mL tube at 350 x g for 5min at 4 °C.
5. 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.
6. Aliquot 5 x 10⁶ BM cells into a new 15 mL tube for CyTOF staining.
7. Spin the 15 mL tube aliquot at 350 x g for 5 min at 4 °C.
8. 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.
9. After incubation, add 4 mL of CyTOF staining buffer to the tube. Spin the tube at 350 x g for 5 min at 4 °C. For human BM, add 10% human AB serum into the CyTOF staining buffer.
10. Carefully aspirate the supernatant and resuspend the BM cells with 50 µL of Fc Receptor blocking solution. Incubate for 10 min at 4 °C. Skip this step for human BM.
11. Add 50 µL of the homemade CyTOF antibody cocktail⁵ to the sample so the total staining volume is 100 μL. Gently pipette to mix. Incubate for 30 min at 4 °C. The final volume of the antibody cocktail is 100 μL for both mouse BM and human BM samples.
12. 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 °C.
13. Repeat step 2.12 for a total of two washes.
14. 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.
15. Carefully aspirate the supernatant and resuspend the pellet with 1 mL fresh 1.6% FA solution. Incubate for 15 min at RT.
16. Spin the tube at 800 x g for 5 min at 4 °C.
17. Carefully aspirate the supernatant and resuspend the cell pellet with 125 nM intercalation solution in 1 mL fix/perm buffer.
18. Incubate the sample in intercalation solution overnight at 4 °C.

3. Prepare Cells for CyTOF Acquisition

1. Gently vortex and spin cells at 800 x g for 5 min at 4 °C.
2. Wash cells by adding 2 mL of CyTOF staining buffer, spin cells at 800 x g for 5 min at 4 °C and remove the supernatant by aspiration.
3. Resuspend cells in 1 mL of diH₂O. Reserve a small volume (approximately 10 µL) from each tube to count cells.
4. Spin cells at 800 x g for 5 min at 4 °C.
5. Repeat step 3.3 and 3.4.
6. 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.

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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 heterogeneity^1,2,3. 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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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