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 genera....

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....

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

List of materials used in this article

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.