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

Summary

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.

Abstract

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.

Introduction

Neutrophils are the most abundant leukocytes in humans. They are the main cellular component of the innate immune system and act as a first line of defense against invading microorganisms. Patients with acquired neutropenia and primary immunodeficiencies that affect neutrophil numbers and/or function develop life-threatening invasive bacterial and fungal infections, highlighting the importance of these cells in host defense ¹. Immune recognition of invading pathogens at the infection site by their cognate pattern-recognition receptors results in the induction of an orchestrated innate immune response, which leads to secretion of chemoattractants that generate a chemotactic gradient capable of recruiting neutrophils from the bloodstream into the inflamed tissue ². After neutrophils enter the infection site, they become activated, which leads to cytokine and chemokine production, pathogen uptake, and killing via oxidative and non-oxidative mechanisms ³. Besides their well-recognized role in innate immunity, neutrophils have also been recently shown to play important roles as initiators of effective adaptive immune responses ⁴. On the other hand, apart from their protective roles in immunity, neutrophils may also mediate tissue injury and immunopathology due to excessive accumulation and/or activation at sites of inflammation, as shown in a variety of infectious and autoimmune diseases ^5-7.

Despite the indispensable role of neutrophils in mounting effective innate immune responses and their pleiotropic effector functions in several infectious diseases and inflammatory conditions, technical difficulties with handling these cells and lack of reliable experimental protocols has hindered research with neutrophils over the past decades. Therefore, use of reproducible assays for isolation of neutrophils should facilitate further research on neutrophil-mediated immunological functions ex vivo and in vivo. To date, several methods have been described for the isolation of neutrophils such as density gradient centrifugation of human blood and mouse blood or bone marrow ^8,9, positive or negative immunomagnetic enrichment of neutrophils from mouse blood or bone marrow ^10,11, and harvesting of neutrophils from the peritoneal cavity of mice following intraperitoneal injection of thioglycollate or other inflammatory agents ¹². Although neutrophils can be easily isolated in large numbers from human blood, this method is suboptimal in mice due to the limited volume of mouse blood that precludes isolation of sufficient neutrophils for functional studies or adoptive transfer experiments ¹³. In addition, although the yield of thioglycollate-elicited cells from the peritoneal cavity is greater compared to that of mouse blood, the purity of neutrophils in the inflammatory peritoneal lavage varies between 60-90%, and the isolated neutrophils exhibit an activated phenotype. Thus, the cells collected using this method can only be used for performing functional studies of activated but not of unstimulated neutrophils, as the mouse peritoneal cavity has few neutrophils at the steady state ¹². Instead, the bone marrow is a convenient reservoir for harvesting large numbers of either unstimulated or activated neutrophils ^11,14, which can then be used for downstream functional studies such as phagocytosis, killing and degranulation, or for adoptive transfer into recipient mice.

Herein we describe a simple and fast (~ 2 hr) protocol, which provides a high yield (~6-12 × 10⁶ neutrophils/uninfected mouse, or up to 30-40 × 10⁶ neutrophils/infected mouse) of pure (80-95%) neutrophils with >95% viability from the bone marrow. This method uses commercially available Histopaque, which are density gradient cell separation media consisting of Ficoll and sodium diatrizoate, to separate neutrophils from the bone marrow of mice. This method yields significantly larger numbers of neutrophils per mouse compared to blood or peritoneal cavity, it can be used to collect neutrophils from mice both at steady state or after infection, and it is easier to layer compared to the density gradient centrifugation method that uses discontinuous Percoll gradients consisting of 55%/65%/75% Percoll in PBS ⁹. In addition, the time and resources required to collect pure neutrophils are significantly decreased compared to neutrophil isolation using Fluorescence-Activated Cell Sorting. Also, because this method does not involve an immunomagnetic enrichment step, it is more cost-effective, and it avoids the exposure of cells to the magnetic column and antibodies, thus decreasing the likelihood of neutrophil activation.

In addition to performing functional studies of isolated neutrophils ex vivo and adoptive transfer of cells into recipient mice, this protocol also describes a method for labeling of isolated neutrophils using different CellTracker dyes. Differential labeling of neutrophils from mice of various genetic backgrounds can be adapted in competitive repopulation studies for tracking the transferred neutrophils in tissues of recipient mice using flow cytometry, which can provide mechanistic insight on the direct role of specific genes in trafficking of neutrophils from the blood into target inflamed organs ⁶.

Protocol

1. Isolation of Mouse Bone Marrow Cells

1. Euthanize mice using the institution's animal care committee-approved protocol and spray the animal surface with 70% ethanol.
2. Make an incision of the skin in the mid-abdomen and remove the skin from the distal part of the mouse including the skin covering the lower extremities.
3. Cut off the muscles from the lower extremities using scissors and carefully dislocate the acetabulum from the hip joint, while avoiding breaking the femur head.
4. Remove the remaining muscles from the femur and tibia using a scalpel and scissors and separate the femur from the tibia at the knee joint exercising care to not break the bone ends. Place the bones in a Petri dish containing ice-cold RPMI 1640 1X supplemented with 10% FBS and 1% Penicillin/streptomycin.
5. Proceed to the following steps under a tissue culture hood. Take extra precaution to maintain strict sterile techniques to avoid neutrophil activation.
6. Rinse each bone with 70% ethanol (within a Petri dish) followed by three subsequent washes in ice-cold sterile PBS (within Petri dishes) to rinse off the ethanol from the surface of the bones.
7. Inside a clean sterile Petri dish, cut off the epiphyses of the bones and keep them aside.
8. Use a 25-gauge needle and a 12 cc syringe filled with RPMI supplemented with 10% FBS and 2 mM EDTA, and flush the bone marrow cells from both ends of the bone shafts onto a 50 ml screw top Falcon tube fitted with a 100 μm filter. In order to efficiently remove all cells, scrape the inner surface of the bones using the 25-gauge needle.

NOTE: Blanching of bones indicates that the cells have been sufficiently scraped.

NOTE: Use approximately 10 ml of media to flush a femur/tibia pair. Adding EDTA to the medium is essential to prevent clumping of the cells.

9. Cut the bone epiphyses in small 0.5-1 mm³ pieces with a scalpel and smash them through the 100 μm filter using the back end of a 2.5 ml Eppendorf Combitip Plus Biopur pipette tip.
10. Centrifuge at 1,400 rpm for 7 min at 4 °C.
11. Lyse the red blood cells by resuspending the cell pellet in 20 ml of 0.2% NaCl for approximately 20 sec followed by addition of 20 ml of 1.6% NaCl. (Critical: Do not exceed 20-30 sec of hypotonic lysis to avoid bone marrow cell death. The use of hypotonic NaCl for lysis is recommended over ACK lysing buffer because the latter has the potential to activate the neutrophils).
12. Centrifuge for 7 min at 1,400 rpm at 4 °C to collect the cells.
13. Wash cells with RPMI 1640 1X supplemented with 10% FBS and 2 mM EDTA and centrifuge as in step 1.12.
14. The yield of bone marrow cells using this method is approximately 60-80 million per uninfected 8-12 week-old C57BL/6 mouse.

2. Separation of Neutrophils by Density Gradient Centrifugation

1. Count the bone marrow cells and resuspend in 1 ml of ice-cold sterile PBS.
2. Add 3 ml of Histopaque 1119 (density, 1.119 g/ml) in a 15-ml conical tube.
3. Overlay 3 ml of Histopaque 1077 (density, 1.077 g/ml) on the 3 ml of Histopaque 1119.

NOTE: Histopaque 1119 and Histopaque 1077 should be warmed to 18-26 °C before use.

Critical Step: Prepare gradients immediately before use as preparing the gradient in advance will result in diffusion between the two layers and suboptimal neutrophil purity and recovery.

Critical Step: Overlaying Histopaque 1077 over 1119 needs to be done slowly in order to avoid mixing the two densities, which will preclude cell separation during centrifugation.

4. Overlay the bone marrow cell suspension on top of the Histopaque 1077.

Critical Step: Overlaying the bone marrow cell suspension over Histopaque 1077 needs to be done slowly in order to avoid disturbing the interface between the cells and Histopaque 1077.

NOTE: Resuspending bone marrow cells from an uninfected mouse in 1 ml of PBS yields neutrophil purity of >90%. However, pooling many bone marrow samples compromises neutrophil purity; for example, resuspending 300 x 10⁶ cells in 3 ml PBS reduces neutrophil purity from >90% to ~80%. Therefore, investigators should perform pilot experiments to identify the ideal cell count/volume conditions for their specific experiments

5. Centrifuge for 30 min at 2,000 rpm at 25 °C without brake.

NOTE: Centrifugation of the gradient at room temperature is critical and essential for effective separation of the neutrophils.

6. Collect the neutrophils at the interface of the Histopaque 1119 and Histopaque 1077 layers.
7. Wash the collected neutrophils twice with RPMI 1640 1X supplemented with 10% FBS and 1% Penicillin/streptomycin and centrifuge at 1,400 rpm for 7 min at 4 °C.
8. Count the neutrophils and determine their viability.

NOTE: Neutrophils are typically >95% viable and >90% pure as determined by FACS analysis. The typical yield of neutrophils from the bone marrow (i.e. 2 femur and 2 tibia bones) of an uninfected 8-12 week-old C57BL/6 mouse is ~6-12 million cells. This number is substantially greater when neutrophils are harvested from bone marrow of infected animals. Hence, ~30-40 million neutrophils/mouse were recovered when Candida-infected mice were used for cell harvesting ⁶.

3. Labeling of Neutrophils Using CellTracker Dyes

1. Resuspend the neutrophils at 5 x 10⁶ cells/ml in PBS prewarmed at 37 °C.
2. Add a CellTracker dye at a final concentration of 5 μM.

NOTE: CellTracker Green (CMFDA (5-Chloromethylfluorescein Diacetate) and CellTracker Orange (CMTMR (5-(and-6)-4-Chloromethyl Benzoyl Amino Tetramethylrhodamine) was used in this protocol to differentially label neutrophils from wild-type and gene-deficient mice.

NOTE: Prepare a stock solution of 10 mM of CellTracker Green and CellTracker Orange, aliquot, and store at -80 °C until the day of the experiment.

3. Incubate neutrophils with 5 μM of the corresponding CellTracker dye for 10 min at 37 °C in a shaking water bath in the dark.
4. Wash cells twice with ice-cold RPMI 1640 1X supplemented with 10% FBS and 1% Penicillin/streptomycin.

NOTE: Efficient washing of the cells after the labeling step with the CellTracker dye is essential to avoid dye cross-contamination before mixing differentially-labeled neutrophil populations for downstream competitive repopulation studies.

4. Adoptive Transfer of Neutrophils in Mice and Analysis of Transferred Neutrophils Using Flow Cytometry

1. Resuspend neutrophils in ice-cold PBS at a concentration of 25 x 10⁶ cells/ml and inject 200 μl of the suspension into the lateral tail vein so that 5 x 10⁶ neutrophils are transferred per mouse. For competitive repopulation neutrophil studies, mix wild-type and gene-deficient neutrophils at a 1:1 ratio and inject a total of 5 x 10⁶ neutrophils per mouse as above.

NOTE: At least up to 10 x 10⁶ neutrophils may be adoptively transferred per mouse without obvious immediate toxicity to the animals.

2. At different times following adoptive transfer (e.g. 1, 2, 3 or 4 hr post-transfer), euthanize mice and harvest blood, and/or bone marrow and/or other target organ(s) of interest.
3. Prepare single cell suspensions from these tissues for quantitative and qualitative analysis of adoptively transferred labeled neutrophils using published protocols ^6,15.
4. Following live/dead viability staining and Fc blockade, label cells with CD45 (clone 30-F11), Ly6G (clone 1A8) and CD11b (clone M1/70) and gate on live CD45⁺ Ly6G⁺ CD11b⁺ neutrophils. Neutrophils in this gate include native neutrophils of the recipient mouse as well as the adoptively transferred labeled neutrophils, which are FITC⁺ (if labeled with CellTracker Green) or PE⁺ (if labeled with Cell Tracker Orange).

NOTE: Neutrophils may be tracked in blood, bone marrow and kidney of Candida-infected mice for at least 4 hr post-transfer.

NOTE: Fixation with 2% paraformaldehyde in PBS does not adversely affect the mean fluorescence intensity of the neutrophils labeled with CellTracker Green or CellTracker Orange for at least 48 hr.

Results

This protocol is optimized for the harvest of bone marrow cells from mice and the subsequent separation of neutrophils from these cells by density gradient centrifugation using commercially available Histopaque cell separation media. Neutrophils isolated using this method can be used for a variety of downstream functional studies ex vivo and for adoptive transfer experiments in recipient mice.

The typical yield of collection of bone marrow cells from both femurs and tibia per uninfect...

Discussion

Herein we present a reliable, simple, fast and economical protocol for isolation of large numbers of neutrophils from the bone marrow of mice with high purity and viability using a density gradient centrifugation approach. When this protocol is performed correctly, ~6-12 × 10⁶ neutrophils can be recovered from an uninfected mouse and as many as ~30-40 ×10⁶ neutrophils may be isolated from a mouse after infection ⁶. The isolated neutrophils are 80-95% pure and >95% viable.

Materials

Name	Company	Catalog Number	Comments
			Reagents
RPMI 1640 1X with L-glutamine and 25 mM HEPES	Cellgro	10-041-CV
Fetal Bovine Serum (Heat inactivated)	GemCell	100500
Penicillin/Streptomycin (10,000 units penicillin / 10,000 mg/ml strep)	GIBCO	15140
0.5 M EDTA	Quality Biological Inc	351-027-101
0.2% and 1.6% sodium chloride	JT Baker	3624	Sodium chloride solutions prepared using distilled water and sterile filtered
Sterile filtered Histopaque 1077	Sigma	10771	Histopaque 1077 needs to be brought to 18-26 °C before use
Sterile filtered Histopaque 1119	Sigma	11191	Histopaque 1119 needs to be brought to 18-26 °C before use
Phosphate Buffered Saline (PBS) without Calcium and Magnesium	Cellgro	210-40-CV
Ethyl Alcohol (200 proof)	The Warner Graham Company	64-17-5
CellTracker Green (CMFDA, 5-Chloromethylfluorescein Diacetate)	Invitrogen	C-7025	Make stock solutions of 10 mM in DMSO, aliquot and store at -80 °C
CellTracker Orange (CMTMR, 5-(and 6)-4-Chloromethyl Benzoyl Amino Tetramethylrhodamine)	Invitrogen	C-2927	Make stock solutions of 10 mM in DMSO, aliquot and store at -80 °C
Anti-mouse CD45 (Clone 30-F11)	eBioscience	170451-82
Anti-mouse Ly6G (Clone 1A8)	BD Pharmingen	551461
Anti-mouse Ly6G (Clone 1A8)	BD Pharmingen	560599
Anti-mouse CD11b (Clone M1/70)	eBioscience	47-0112-82
Anti-mouse CD16/CD32 (Mouse BD Fc Block)	BD Pharmingen	553141	Use at 1:100 dilution
LIVE/DEAD Fixable Blue Dead Cell Stain Kit	Molecular Probes	L-23105	Use at 1:1000 dilution
Dimethyl Sulfoxide (DMSO)	Sigma	67-68-5
Paraformaldehyde Solution, 4% in PBS	USB Corporation	19943
			Table 1. Reagents for isolation, labeling and tracking of adoptively transferred neutrophils.
			Materials
C57BL/6 mice	Taconic
15 ml centrifuge tubes	Corning	430053
50 ml centrifuge tubes	BD Falcon	352070
25 ml serological pipettes	Celltreat	229225B
10 ml serological pipettes	Celltreat	229210B
5 ml serological pipettes	Celltreat	229205B
Pasteur pipettes (3 ml)	BD Falcon	357575
12 ml syringes	Kendall monoject	512878
25 G x 5/8 in. Needles (precision glide needles)	BD	305122
100 mm cell strainers	BD Falcon	352360
Bactericidal Petri dishes	BD Falcon	351029
Combitips Plus Biopur	Eppendorf	2249608-5
Mouse dissecting instruments (Scissors, forceps, scalpel)	Biomedical Research Instruments	10-2300, 10-2165, 25-1200, 26-1000	Instruments sterilized prior to use
			Equipment
Tissue culture hood	The Baker Company	SG403
Refrigerated centrifuge	Thermo Fischer Scientific	75004521
37 °C shaking water bath	Thermo Fischer Scientific	3166721
			Table 2. List of Materials and Equipment used in this protocol.