Isolation of Mouse Neutrophils

Abstract

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes reproducible density gradient centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments.

INTRODUCTION

Neutrophils comprise the main cellular component of the innate immune system and the first line of defense against pathogens (de Oliveira et al., 2016; Desai and Lionakis, 2018; Lionakis et al., 2023). Immune recognition of the invading pathogen triggers a local innate immune response that results in the prompt expansion of neutrophils in the bone marrow and their recruitment to the site of infection. Once mobilized at the infected tissue, neutrophils become activated and mediate a variety of effector functions including binding, uptake and killing of pathogens by oxidative and/or non-oxidative mechanisms, degranulation of proteases in the extracellular space and production of pro-inflammatory and anti-inflammatory mediators that shape and modulate the outcome of the innate and adaptive antimicrobial immune response (Amulic et al., 2012). On the other hand, some of these neutrophil functions, when in excess or dysregulated, may promote tissue injury and immunopathology instead of appropriate antimicrobial activity (Kruger et al., 2015; Narasaraju et al., 2011; Soehnlein et al., 2017). Therefore, continued research is needed to elucidate the molecular mechanisms by which neutrophils induce protective or detrimental immune responses in the context of various infectious and non-infectious inflammatory conditions (Hedrick and Malanchi, 2022; Sas et al., 2020).

This unit describes methods for isolating and enriching neutrophils from mouse bone marrow or various mouse tissues. Basic Protocol 1 describes a protocol for harvesting large numbers of neutrophils from the bone marrow of mice both at steady state and under inflammatory conditions followed by purification by positive immunomagnetic selection that relies on Ly6G, a mouse neutrophil-specific cell surface marker. The inflammatory condition is achieved by infecting mice with Candida albicans although other pathogens can be used to suite experimental conditions. Neutrophils can also be purified from mouse bone marrow by negative immunomagnetic separation (Alternate Protocol 1) or by density gradient centrifugation method (Alternate Protocol 2). Basic Protocol 2 describes a method for harvesting of neutrophils from mouse kidney, liver or spleen followed by purification by positive immunomagnetic selection. Tissue neutrophils can also be enriched by FACS using antibodies targeted against surface expression of CD45, Ly6G, and CD11b (Alternate Protocol 3). The number and purity of neutrophils obtained from these protocols are sufficient for investigating neutrophil phagocytosis, intracellular and extracellular killing, oxidative burst, chemotaxis, degranulation, survival, cytokine and chemokine production; for performing transcriptional profiling of isolated neutrophils; for adoptive transfer experiments of isolated neutrophils into recipient mice; and for labeling of neutrophils with dyes and tracking their trafficking in various tissues in vivo following adoptive transfer into recipient mice.

NOTE: Protocols using live animals must fırst be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.

BASIC PROTOCOL 1

ISOLATION OF NEUTROPHILS FROM MOUSE BONE MARROW USING POSITIVE IMMUNOMAGNETIC SEPARATION

This protocol describes a technique to isolate cells from the bone marrow of naïve or infected mice followed by purification of neutrophils using positive immunomagnetic selection. Neutrophils can also be purified from the bone marrow using negative immunomagnetic separation (Alternate protocol 1) or by using density gradient centrifugation method (Alternate protocol 2).

Materials

C57BL/6 mice

Ice

Refrigerated and room temperature centrifuges

Tissue culture hood

Additional reagents and equipment for euthanasia of mice, for optional infection of mice, and for counting viable cells by trypan blue exclusion

70% (v/v) ethanol solution

1 × RPMI 1640 with l-glutamine and 25 mM HEPES (Corning, cat. no. 10-041-CV)

Heat-inactivated fetal bovine serum (FBS) (Gemini Bioproducts, cat. no. 100-106)

Bovine serum albumin (BSA) (Fisher Scientific, cat. no. BP9705-100)

Penicillin/streptomycin (10,000 U penicillin/10,000 μg/ml streptomycin) (Corning, cat. no. 30-002-CI)

Phosphate-buffered saline (PBS) 1x without calcium and magnesium (Corning, cat. no. 21-040-CV)

Hanks-Balanced Salt Solution (HBSS) 1x without calcium, magnesium and phenol red (Corning, cat. no. 21-022-CV)

EDTA (Corning, cat. no. 46034-CI)

FACS buffer: Phosphate-buffered saline (PBS) (Corning, cat. no. 21-040-CV) without calcium and magnesium supplemented with 0.5% bovine serum albumin (BSA) (Fisher Scientific, cat. no. BP9705-100) and 0.01% sodium azide [10% (w/v) sodium azide (Teknova, cat. no. S0209)]

ACK lysis buffer (Quality Biological, cat. no.118-156-721)

Surgical instruments (kept in 70% ethanol solution) including forceps, scalpels, and scissors

25-G × 5/8-in. needles (BD, cat. no. 305122)

1-ml Monoject^™ tuberculin syringe (Covidien, cat. no. 8881501400)

20-ml Monoject^™ luer-lock syringe (Covidien, cat. no. 8881520657)

100 × 15–mm petri dishes (Corning, cat. no. 351029)

FACS tubes (BD Biosciences, cat. no. 352053)

15-ml conical centrifuge tubes (Corning, cat. no. 430053)

50-ml conical centrifuge tubes (Corning, cat. no. 352098)

100-μm cell strainers (Corning, cat. no. 352360)

3-ml Pasteur pipets (Corning, cat. no. 357575)

10-ml serological pipets (Corning, cat. no. 356551)

25-ml serological pipets (Corning, cat. no. 356525)

Anti-Ly6G MicroBeads Ultrapure for mouse neutrophils (Miltenyi, cat. no. 130-120-337)

Magnetic-activated cell sorting (MACS) buffer: PBS (Corning, cat. no. 21-040-CV) containing 2 mM EDTA (Corning, cat. no. 46034-CI) and 0.5% bovine serum albumin (BSA) (Fisher Scientific, cat. no. BP9705-100)

LS+ positive selection columns (Miltenyi, cat. no. 130-042-401)

QuadroMACS Separator (Miltenyi, cat. no. 130-090-976)

MACS MultiStand (Miltenyi, cat. no. 130-042-303)

Trypan Blue 0.4% in 0.85% NaCl (Lonza, cat. no. 17-942E)

LIVE/DEAD fixable blue dead cell stain kit (Invitrogen, cat. no. L-23105)

Anti-mouse CD45 APC (clone 30-F11; BD Biosciences, cat. no. 559864)

Anti-mouse Ly6G PE (clone 1A8; BD Biosciences, cat. no. 551481)

Anti-mouse CD11b APC-eFluor^® 780 (clone M1/70; eBioscience, cat. no. 47-0112-82)

Anti-mouse CD16/CD32 (clone 93; eBioscience, cat. no. 14-0641-86)

Additional reagents and equipment for flow cytometry

NOTE: All prepared solutions can be sterile-filtered using 0.2-μm filters and stored at 4°C.

Harvest bone marrow cells from mouse femurs and tibias

• 1Euthanize control mice using an approved IACUC procedure and spray the animal skin with 70% ethanol.
• 2Using scissors, make an incision in the mid-abdomen on the ventral side and remove the skin to expose the abdomen and lower extremities.
• 3
  Remove the muscles from both legs using scissors and carefully dislocate the acetabulum from the hip joint.

  Exercise caution to avoid breaking the femur head.

• 4After separating the bones perform all subsequent steps under the tissue culture hood to prevent contamination and neutrophil activation. Place bones in a 100 × 15–mm sterile petri dish containing 15 ml ice-cold 1 × RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Keep the petri dish on ice.
• 5
  Further remove the remaining muscles from the femurs and tibias using a scalpel and separate the femur from the tibia at the knee joint.

  Exercise caution to avoid breaking the femur and tibia bone ends while separating them.

• 6Sterilize each bone by rinsing in a 70% ethanol solution inside a petri dish followed by three subsequent washes in 15 ml ice-cold sterile PBS within the corresponding petri dishes to rinse off the ethanol from the surface of the bones.
• 7Using a scalpel, cut off the epiphyses of the bones and keep them aside in an empty sterile petri dish.
• 8
  Using a 25-G × 5/8-in. needle and a 20-ml syringe filled with PBS supplemented with 2 mM EDTA, flush the bone marrow cells into a 50-ml conical tube through a 100-μm cell strainer.

  EDTA is used to avoid blood clotting and cell clumping.

  Blanching of bones typically indicates that the bones have been sufficiently scraped to provide a good yield of bone marrow cells. Approximately 5 ml of PBS supplemented with 2 mM EDTA is required to flush each femur and tibia pair.

• 9Cut the bone epiphyses in small 0.5- to 1-mm³ pieces with a scalpel and mash them through the 100-μm cell strainer using the rubber end of the syringe plunger insert of a 1-ml tuberculin syringe. Spray surface of filter with 5 ml PBS supplemented with 2 mM EDTA during the mashing to collect cells in solution below.
• 10Pellet the bone marrow cells by centrifuging for 7 min at 425 × g, 4°C.
• 11To lyse the red blood cells, resuspend the cell pellet in 5 ml of ACK lysis buffer using a 10-ml serological pipet and pipette up and down slowly for 30 sec followed by addition of 25 ml of HBSS supplemented with 2mM EDTA to stop the reaction.
• 12Centrifuge for 7 min at 425 × g, 4°C, to collect the bone marrow cells.
• 13Wash the cells with 10 ml of 1 × RPMI 1640 supplemented with 10% FBS and 2 mM EDTA using a 10 ml serological pipet and centrifuge again as in step 12.

Purify neutrophils using Ly6G-based positive immunomagnetic selection

• 14Count the cells and resuspend cell pellet in 90 μl MACS buffer per 10⁷ cells.
• 15Add 10 μl of anti-Ly6G microbeads per 10⁷ cells.
• 16
  Mix well with a pipet and incubate for 10 min in a refrigerator (2° - 8°C).

  Do not incubate on ice.

• 17Add 1-2 ml MACS buffer per 10⁷ cells in each sample.
• 18Centrifuge for 10 min at 300 × g, 4°C.
• 19
  Aspirate the supernatant completely and resuspend in 500 μl MACS buffer.

  If >10⁸ cells are to be used, adjustments are required in the amount of buffer added per the manufacturer’s instructions.

• 20
  Place a LS column per sample on the magnet.

  Similar results with high neutrophil numbers, purity and viability are obtained by using LS columns and a QuadroMACS separator, as well as by using autoMACS columns and an autoMACS or an autoMACS Pro separator.

• 21
  Rinse with 3 ml MACS buffer per column and discard the flow through.

  Avoid adding bubbles to the column as this will block flow through.

• 22Apply the cell suspension to the column.
• 23Wash the column three times, each time with 3 ml MACS buffer.
• 24Remove the column from the magnet and place onto a new 15-ml conical centrifuge tube.
• 25Add 5 ml MACS buffer to the column and immediately flush the cells with the plunger provided along with the column.
• 26Count neutrophils with trypan blue exclusion method to measure yield and viability and take an aliquot for flow cytometry analysis to also measure viability with a fluorescent viability dye and purity by staining with APC-conjugated anti-CD45, PE-conjugated anti-Ly6G, and APC-eFluor^® 780–conjugated anti-CD11b.

ALTERNATE PROTOCOL 1

PURIFICATION OF NEUTROPHILS FROM BONE MARROW USING NEGATIVE IMMUNOMAGNETIC SEPARATION

As an alternative to Ly6G-based positive immunomagnetic selection, bone marrow neutrophils can also be enriched by negative immunomagnetic separation.

Additional Materials (also see Basic Protocol 1)

Neutrophil Isolation Kit for mouse neutrophils (Miltenyi, cat. no. 130-097-658)

Purify neutrophils using negative immunomagnetic separation

1. Following harvest of bone marrow cells from mouse femurs and tibias (see Basic Protocol 1 steps 1 – 13), count the cells and resuspend cell pellet in 50 μl MACS buffer per 10⁷ cells.

2. Add 10 μl of Neutrophil Biotin-Antibody Cocktail per 10⁷ cells.

3. Mix well with a pipet and incubate for 10 min in a refrigerator (2° - 8°C).

   Do not incubate on ice.

4. Add 1-2 ml MACS buffer per 10⁷ cells in each sample.

5. Centrifuge for 10 min at 300 × g, 4°C.

6. Aspirate the supernatant completely and resuspend in 80 μl MACS buffer per 10⁷ cells.

7. Add 20 μl of Anti-Biotin Microbeads per 10⁷ cells.

8. Mix well with a pipet and incubate for 15 min in a refrigerator (2° - 8°C).

   Do not incubate on ice.

9. Add 1-2 ml MACS buffer per 10⁷ cells in each sample.

10. Centrifuge for 10 min at 300 × g, 4°C.

11. Aspirate the supernatant completely and resuspend in 500 μl MACS buffer.

    If >10⁸ cells are to be used, adjustments are required in the amount of buffer added per the manufacturer’s instructions.

12. Place a LS column per sample on the magnet.

    Similar results with high neutrophil numbers, purity and viability are obtained by using LS columns and a QuadroMACS separator, as well as by using autoMACS columns and an autoMACS or an autoMACS Pro separator.

13. Rinse with 3 ml MACS buffer per column and discard the flow through.

    Avoid adding bubbles to column as this will block flow through.

14. Apply the cell suspension to the column and collect the flow through containing unbound neutrophils.

15. Wash the column three times, each time with 3 ml MACS buffer and collect all flow through.

16. Count neutrophils with trypan blue exclusion method to measure yield and viability and take an aliquot for flow cytometry analysis to also measure viability with a fluorescent viability dye and purity by staining with APC-conjugated anti-CD45, PE-conjugated anti-Ly6G, and APC-eFluor^® 780-conjugated anti-CD11b.

ALTERNATE PROTOCOL 2

PURIFICATION OF NEUTROPHILS FROM BONE MARROW USING HISTOPAQUE-BASED DENSITY GRADIENT CENTRIFUGATION

As an alternative to Ly6G-based positive immunomagnetic selection or negative immunomagnetic separation, bone marrow neutrophils can also be enriched by density gradient centrifugation method.

Additional Materials (also see Basic Protocol 1)

Sterile Histopaque 1077 (Sigma-Aldrich, cat. no. 10771)

Sterile Histopaque 1119 (Sigma-Aldrich, cat. no. 11191)

Purify neutrophils using Histopaque-based density gradient centrifugation

1. Following harvest of bone marrow cells from mouse femurs and tibias (see Basic Protocol 1 steps 1 – 13), count the number of viable cells by trypan blue dye exclusion and resuspend in 1 to 3 ml of ice-cold sterile PBS.

   Resuspend cells in 1 ml if harvesting bone marrow from one uninfected mouse. Resuspend cells in 3 ml if harvesting bone marrow from more than one uninfected mouse or from infected mice. Investigators should perform pilot experiments tailored to the specific conditions used, as the density of bone marrow cells can affect neutrophil purity after centrifugation.

2. Add 3 ml of Histopaque 1119 (density, 1.119 g/ml) in a 15-ml conical centrifuge tube.

   Histopaque 1119 should be brought to room temperature before use.

3. Overlay with 3 ml of Histopaque 1077 (density, 1.077 g/ml).

   Exercise caution to avoid disturbing the interface between Histopaque 1119 and Histopaque 1077.

   Histopaque 1077 should be brought to room temperature before use.

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

   Exercise caution to avoid disturbing the interface between the cell suspension and Histopaque 1077.

5. Centrifuge for 30 min at 872 × g, room temperature, without brake.

6. Collect the neutrophils at the interface of the Histopaque 1119 and Histopaque 1077 layers using a 3-ml Pasteur pipet.

7. Wash the collected neutrophils twice with 10 ml 1 × RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin and centrifuge for 7 min at 425 × g, 4°C.

8. Count neutrophils with trypan blue exclusion method to measure yield and viability and take an aliquot for flow cytometry analysis to also measure viability with a fluorescent viability dye and purity by staining with APC-conjugated anti-CD45, PE-conjugated anti-Ly6G and APC-Cy7–conjugated anti-CD11b (Lionakis et al., 2011).

BASIC PROTOCOL 2

ISOLATION OF NEUTROPHILS FROM MOUSE TISSUES USING POSITIVE IMMUNOMAGNETIC SEPARATION

Under inflammatory conditions, neutrophils traffic to tissue and acquire a different phenotype relative to the neutrophils produced in the bone marrow, which is modulated by the local inflammatory milieu. This protocol describes a technique to isolate neutrophils from various tissues (kidney, liver, or spleen) of mice using gentle enzymatic digestion of tissue and subsequent positive immunomagnetic selection.

Materials

C57BL/6 mice

Ice

Refrigerated and room temperature centrifuges

Tissue culture hood

Additional reagents and equipment for euthanasia of mice, for optional infection of mice, and for counting viable cells by trypan blue exclusion

70% (v/v) ethanol solution

1 × RPMI 1640 with l-glutamine and 25 mM HEPES (Corning, cat. no. 10-041-CV)

Heat-inactivated fetal bovine serum (FBS) (Gemini Bioproducts, cat. no. 100-106)

Bovine serum albumin (BSA) (Fisher Scientific, cat. no. BP9705-100)

Penicillin/streptomycin (10,000 U penicillin/10,000 μg/ml streptomycin) (Corning, cat. no. 30-002-CI)

Phosphate-buffered saline (PBS) 1x without calcium and magnesium (Corning, cat. no. 21-040-CV)

Hanks-Balanced Salt Solution (HBSS) 1x without calcium, magnesium and phenol red (Corning, cat. no. 21-022-CV)

EDTA (Corning, cat. no. 46034-CI)

FACS buffer: Phosphate-buffered saline (PBS) (Corning, cat. no. 21-040-CV) without calcium and magnesium supplemented with 0.5% bovine serum albumin (BSA) (Fisher Scientific, cat. no. BP9705-100) and 0.01% sodium azide [10% (w/v) sodium azide (Teknova, cat. no. S0209)]

ACK lysis buffer (Quality Biological, cat. no.118-156-721)

Surgical instruments (kept in 70% ethanol solution) including forceps, scalpels and scissors

25-G × 5/8-in. needles (BD, cat. no. 305122)

20-ml Monoject^™ luer-lock syringe (Covidien, cat. no. 8881520657)

100 × 15–mm petri dishes (Corning, cat. no. 351029)

FACS tubes (BD Biosciences, cat. no. 352053)

15-ml conical centrifuge tubes (Corning, cat. no. 430053)

50-ml conical centrifuge tubes (Corning, cat. no. 352098)

100-μm cell strainers (Corning, cat. no. 352360)

3-ml Pasteur pipets (Corning, cat. no. 357575)

10-ml serological pipets (Corning, cat. no. 356551)

25-ml serological pipets (Corning, cat. no. 356525)

Trypan Blue 0.4% in 0.85% NaCl (Lonza, cat. no. 17-942E)

LIVE/DEAD fixable blue dead cell stain kit (Invitrogen, cat. no. L-23105)

Anti-mouse CD45 APC (clone 30-F11; BD Biosciences, cat. no. 559864)

Anti-mouse Ly6G PE (clone 1A8; BD Biosciences, cat. no. 551481)

Anti-mouse CD11b APC-eFluor^® 780 (clone M1/70; eBioscience, cat. no. 47-0112-82)

Anti-mouse CD16/CD32 (clone 93; eBioscience, cat. no. 14-0641-86)

Additional reagents and equipment for flow cytometry

10 × phosphate-buffered saline (PBS) (Corning, cat. no. 46-013-CM)

RPMI (Corning, cat. no. 10-041-CV) without serum

Liberase TL (Roche, cat. no. 05401020001)

DNase I (Roche, cat. no. 10104159001)

Percoll (GE Healthcare, cat. no. 17-0891-01)

37°C water bath

40-μm cell strainers (Corning, cat. no. 352340)

70-μm cell strainers (Corning, cat. no. 352350)

Harvest cells from mouse kidney, liver or spleen as a single cell suspension

1. Anesthetize the mouse after infection using an approved IACUC procedure and perfuse mice with PBS to remove circulating blood cells from the organs of interest.

2. Using scissors, excise the organ of interest and place it in 10 ml RPMI supplemented with 10% FBS and 1% penicillin/streptomycin on ice until ready to use.

3. Place the organ of interest in a 100 x 15-mm petri dish and perfuse with 10 ml of digesting solution (RPMI without serum containing Liberase TL and DNase I) using a 20-ml syringe fitted with a 25-G × 5/8-in. needle.

   The concentration of Liberase TL in the digesting solution is 0.2125 mg/ml for kidney and liver and 0.125 mg/ml for spleen; the concentration of DNase I in the digesting solution is 0.1 mg/ml for all organs (Lionakis et al., 2012).

   Liberase TL is deactivated in the presence of serum.

4. Finely mince the organ of interest into 1-mm³ size pieces using a scalpel and collect them in a 50-ml conical centrifuge tube using a 10-ml serological pipet.

   A total of 10 ml of digesting solution is required for kidney and spleen digestion, while 20 ml of digesting solution is needed for liver digestion.

5. Incubate the organs at 37°C in a shaking water bath with digesting solution for 20 min.

   Longer digestion times result in decreases in cell viability.

6. At the end of the 20 min incubation, add an equal volume of RPMI with 10% FBS (i.e., 10 ml for kidney/spleen and 20 ml for liver) using a 25-ml serological pipet and place the 50-ml conical centrifuge tube on ice to stop the digestion.

   Both addition of serum and placement of tube on ice inhibit the activity of Liberase TL.

7. Pass the digested tissue through a cell strainer.

   Use a 70-μm cell strainer for kidney and liver; 100-μm cell strainer for spleen.

8. Centrifuge for 7 min at 425 × g, 4°C, to collect the cells.

9. Lyse the RBCs by incubating in 5 ml ACK lysing buffer for 30 to 40 sec and mixing up and down five times with a 10-ml serological pipet. At the end of 30 to 40 sec, add cold HBSS (without calcium and magnesium) with 2 mM EDTA and pass it through the 40-μm filter.

10. Wash the cells with 20 ml FACS buffer and collect cells by centrifuging for 7 min at 425 × g, 4°C. The spleen cells are now ready for purification of neutrophils by magnetic or FACS separation. For liver and kidney cells, continue through density centrifugation steps 11 - 13 as a requirement to obtain a single cell suspension.

11. Resuspend the kidney or liver cells in 8 ml of 40% Percoll and overlay them onto 3 ml of 70% Percoll in a 15-ml conical centrifuge tube using a 3-ml Pasteur pipet.

    40% and 70% Percoll dilutions are made from a “100% Percoll” solution using FACS buffer. The “100% Percoll” solution is generated by adding 5 ml of 10 × PBS in 45 ml FACS buffer.

    Exercise caution to avoid disturbing the interface between 40% and 70% Percoll solutions.

12. Centrifuge for 30 min at 872 × g, without brake at room temperature.

13. Harvest the cells from the 70%/40% Percoll interface, wash twice with 10 ml FACS buffer and resuspend in MACS or FACS buffer (1 × 10⁶ cells/100 μl) for proceeding with magnetic separation or FACS sorting of neutrophils, respectively.

Purify neutrophils using Ly6G-based positive immunomagnetic selection

1. Follow Basic Protocol 1, steps 14 - 26

ALTERNATE PROTOCOL 3

ISOLATION OF NEUTROPHILS FROM MOUSE TISSUES USING FACS

As an alternative to Ly6G-based positive immunomagnetic selection, tissue neutrophils can also be enriched by FACS using antibodies targeted against surface expression of CD45, Ly6G and CD11b.

Additional Materials (also see Basic Protocol 1 if isolating from bone marrow or Basic Protocol 2 if isolating from tissues)

Sorting buffer: PBS without calcium and magnesium (Corning, cat. no. 21-040-CV) supplemented with 10% FBS (Gemini Bioproducts, cat. no. 100-106) and 0.5 mM EDTA (Corning, cat. no. 46034-CI)

Purify neutrophils using FACS

1. Count the cells and resuspend in FACS buffer (PBS + 0.5% BSA + 0.01% sodium azide) at 4°C at a concentration of 1 × 10⁷ cells/ml in a sterile FACS tube (5-ml polystyrene round-bottom tube).

2. Proceed with Fc-blockade by adding anti-CD16/CD32 and incubating for 15 min at 4°C in the dark.

   The optimal amount of antibody required is ~1 μl per 1 × 10⁶ cells.

3. Add APC-conjugated anti-CD45, PE-conjugated anti-Ly6G and APC-eFluor^® 780–conjugated anti-CD11b and incubate for 30 min at 4°C in the dark.

   The optimal amount of antibody required is approximately 1 μl per 1 × 10⁶ cells for each antibody.

4. Wash off excess antibodies by adding 1 ml FACS buffer to each tube and centrifuge for 7 min at 425 × g, 4°C.

5. Repeat the wash and centrifugation step (see step 4).

6. Resuspend the cell pellet at a concentration of 5 × 10⁶ cells/ml in sorting buffer (PBS without calcium and magnesium supplemented with 10% FBS and 0.5 mM EDTA).

7. Sort CD45⁺Ly6G⁺CD11b⁺ cells using standard sorting procedures.

COMMENTARY

Background Information

The mouse bone marrow is a convenient reservoir for isolating large numbers of neutrophils both at steady state under homeostatic conditions and during activation under a variety of infectious and non-infectious inflammatory conditions. It is the most suitable anatomical location in mice from where a large number of neutrophils can be harvested at steady state for immunological assays and adoptive transfer experiments. Bone marrow neutrophils have been shown to be functionally similar to blood neutrophils in mice and have been reported to survive for a longer period of time in culture (Boxio et al., 2004), thus making them a very useful resource for research studies of neutrophil biology and physiology.

Bone marrow neutrophils obtained from uninfected and infected mice can be used for experiments to assess binding, uptake and killing of pathogens by neutrophils intracellularly and extracellularly, to examine neutrophil chemotaxis, survival, degranulation and oxidative burst (Desai et al., 2023). Additional downstream assessments include performing transcriptional analyses of harvested neutrophils (Xie et al., 2020), measuring their potential to secrete cytokines and chemokines, and performing adoptive transfer of cells into recipient mice with the option of neutrophil dye labeling to track neutrophil trafficking in various tissues in vivo (Lionakis et al., 2012).

Importantly, under inflammatory conditions it becomes critical to investigate the effector function of neutrophils from the site of infection as their activity may differ from that of bone marrow neutrophils, driven by factors present in the local immunological microenvironment of the infected tissue (Whitney et al., 2014). In certain other gene-deficient settings, comparative analysis of bone marrow and tissue neutrophils may reveal comparable abnormalities in both compartments (Swamydas et al., 2016). In Basic Protocol 2 we describe a method to isolate neutrophils from mouse kidney, liver or spleen, however this method can be adapted to also allow neutrophil isolation from other mouse tissues. Ly6G is a neutrophil-specific cell surface marker in mice (Daley et al., 2008), which enables Ly6G-based positive selection strategies for high yield, high purity and high viability of neutrophils from mouse tissues under a variety of infectious and non-infectious inflammatory conditions that mimic human disease models in mice.

Critical Parameters and Troubleshooting

Care should be taken to maintain sterile techniques throughout all steps of the described protocols because LPS can activate neutrophils, which once activated can secrete cytokines and undergo apoptosis.

The method we present in Alternate Protocol 2 utilizes Histopaque cell separation media, which consist of sodium diatrizoate and Ficoll. This method is easier to layer compared to the density gradient centrifugation method that utilizes discontinuous Percoll gradients consisting of 55%/65%/75% Percoll in PBS, which often results in intermixing of the Percoll interfaces due to the small density differences between the 55%, 65% and 75% Percoll densities. While performing Histopaque-based density gradient centrifugation, caution should be taken to ensure that Histopaque 1119 and Histopaque 1077 have reached room temperature before overlaying, to maximize neutrophil purity. In addition, the layering of the Histopaque 1119 and Histopaque 1077 gradients should be performed immediately before overlaying the bone marrow cell suspension and not in advance, as after a few minutes two layers intermix. Caution should be also taken when layering Histopaque 1077 onto Histopaque 1119 to avoid intermixing in the Histopaque 1119 and Histopaque 1077 interface. Intermixing of the two densities will significantly decrease the purity of neutrophils. Centrifugation at room temperature, not at 4°C, is essential to maximize purity of neutrophils. Additionally, overlaying more than 100 million bone marrow cells may result in reduction of neutrophil purity from >90% to ~80%. Increasing the volume of PBS for bone marrow cell suspension from 1 ml to 3 ml may ameliorate the decrease in neutrophil purity when large numbers of bone marrow cells are layered over Histopaque 1077. However, it is advised that investigators perform pilot studies tailored to the specific conditions of their experiments to maximize neutrophil purity after Histopaque-based centrifugation.

During magnetic separation, care should be taken to follow the manufacturer’s protocol and to use the appropriate column/magnet separator pair. Incubation with the MACS reagents should be performed in the refrigerator, not on ice, per the manufacturer’s recommendation to avoid non-specific binding of the reagents to cells other than neutrophils, which can adversely affect cell purity. It is important to ensure that the column is emptied between washing steps with MACS buffer and does not contain any residual liquid before proceeding to the next washing step. Conversely, it is important to avoid excessive drying of the column between washing steps with MACS buffer, therefore this step should involve careful monitoring to avoid long pauses once each wash is completed.

Anticipated Results

The typical yield of cells from the bone marrow (i.e., 2 femurs and 2 tibias) of an uninfected 8-12 week-old C57BL/6 mouse is between 60 and 80 million. The typical yield of neutrophils from the bone marrow following positive immunomagnetic selection is ~7-12 million neutrophils with >97% purity and >97% viability as determined by flow cytometry (Fig. 3.20.1A). The typical yield of neutrophils from the bone marrow following negative immunomagnetic separation is ~4-6 million neutrophils with >91% purity and >98% viability (Fig. 3.20.1B). For Histopaque-based gradient centrifugation method for bone marrow neutrophil isolation, the typical yield is is ~6-12 million neutrophils with >91% purity and >98% viability (Fig. 3.20.2).

Figure 3.20.1.

Representative FACS plots depicting purity (left panel) and viability (right panel) of neutrophils isolated from the bone marrow of an uninfected C57BL/6 mouse using A) Ly6G-based positive immunomagnetic selection or B) negative immunomagnetic separation (Neutrophil Isolation Kit).

Figure 3.20.2.

Representative FACS plots depicting purity (left panel) and viability (right panel) of neutrophils isolated from the bone marrow of an uninfected C57BL/6 mouse using the Histopaque-based gradient centrifugation method.

Infection with Candida results in a significant expansion of bone marrow neutrophils, and up to 30 to 40 million neutrophils with high purity (80% to 95%) and high viability (>95%) can be recovered per Candida-infected mouse bone marrow, depending on the infecting inoculum and the timing of bone marrow harvesting post-infection (Lionakis et al., 2012).

Immunomagnetic or FACS-based separation of neutrophils from both kidneys of an 8-12 week old C57BL/6 mouse infected with Candida yields between 1 and 5 million cells, depending on the infecting inoculum and the timing of bone marrow harvesting post-infection. Positive immunomagnetically-enriched neutrophils from infected kidneys are >90% pure and >95% viable, as determined by FACS analysis (Fig. 3.20.3) (Lionakis et al., 2012; Swamydas et al., 2016). FACS-enriched tissue neutrophils are >98% pure and >95% viable.

Figure 3.20.3.

Representative FACS plots depicting purity (left panel) and viability (right panel) of neutrophils isolated from the kidneys of a Candida albicans-infected C57BL/6 mouse using the Ly6G-based positive immunomagnetic selection method.

Time Considerations

Bone marrow cells can be harvested from the femurs and tibias of each mouse in about 15 min. After a single cell suspension has been obtained, an additional 60 min or 90 min are required to obtain enriched neutrophils via positive immunomagnetic selection or negative immunomagnetic separation, respectively. The entire protocol of obtaining enriched neutrophils using Histopaque-based density gradient centrifugation can be completed within 2 hr.

Single cell suspensions from mouse organs can be obtained within 2-4 hr depending on the number of mice used and the organ of interest; for example, harvesting of cells from spleen does not require a Percoll-based centrifugation step following tissue digestion and, hence, it is faster than harvesting of cells from liver or kidney. Once tissue cells are in a single cell suspension, sorting of neutrophils using FACS can be completed within 1 to 2 hr depending on the number of samples and the percent of neutrophils within a given cell suspension.