Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

Summary

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood neutrophils.

Abstract

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

Introduction

Neutrophils constitute a critical subset of leukocytes that play a pivotal role in the innate immune response. They are characterized by multilobed nuclei and granules containing various proteases and antimicrobial peptides¹. Neutrophils primarily function through degranulation, phagocytosis, and the formation of NETs. The observation of NETs was first made by Takei et al. in 1996 during an experiment where neutrophils were stimulated with phorbol myristate acetate (PMA)². Subsequently, the process of NET formation was coined "NETosis" by Brinkmann et al.³ in 2004. Their research further illuminated the crucial role of NETs in neutrophil-mediated antimicrobial responses. NETs are web-like structures composed of chromatin, histones, and antimicrobial proteins that are released from activated neutrophils in response to infectious and inflammatory stimuli. NETs can immobilize and kill invading pathogens by trapping them and exposing them to a high concentration of antimicrobial peptides and proteases^1,3. Additionally, NETs contribute to the clearance of apoptotic cells and participate in inflammation resolution. Recent studies also indicate that an excessive formation of NETs or impaired NET degradation can lead to tissue damage, autoimmune disorders, thrombogenesis, and impaired revascularization^{4,5,6,7,8,9,10}.

The pathogenic role of NETs in uncontrolled fibrosis following myocardial infarction and the formation of ventricular aneurysms has been demonstrated through the expansion of perivascular fibrosis^4,11. The myocardial infarction model and the isolation of neutrophils from bone marrow in mice are both well-established. Polymorphonuclear (PMN) leukocytes, a type of white blood cell abundant in human blood, serve as an excellent source for isolating human neutrophils. This method eliminates the need to harvest bone marrow, thus enhancing safety and efficiency.

NETs also play a role in atrial fibrillation associated with cardiac remodeling. However, large animals such as dogs and pigs were utilized to model atrial fibrillation, as mice lack an atrium sizable enough to establish a re-entrant cycle or the AF model, unless specific ion channels or signaling pathways are knocked down or knocked out¹². While it's possible to induce atrial fibrillation in rats and isolate neutrophils from rat peripheral blood as previously described, researchers encountered a limitation whereby only 2 x 10⁵-5 x 10⁵ neutrophils could be isolated from peripheral blood (10 mL per rat). Extracting sufficient NETs at each time point required approximately 10-25 rats (5 x 10⁶ neutrophils in total), resulting in a time-consuming, expensive, and often low-yield process¹³. In this regard, Li He and colleagues present a bone marrow-oriented strategy to obtain adequate NETs from rats¹⁴. In their article, they provide a comprehensive description of isolating neutrophils from rat bone marrow and compare the NET secretion capabilities of rat peripheral and bone marrow neutrophils. The two methods outlined cater to distinct experimental goals, both resulting in sufficient quantities of rat bone marrow neutrophils while reducing the number of required rats. The two-step isolation method demonstrated superior neutrophil purification, while the one-step method proved time-efficient with acceptable purification levels. Furthermore, the researchers compared NETosis and NET formation between rat bone marrow neutrophils and their peripheral counterparts, finding equal potency with PMN. These findings significantly contribute to neutrophil-related studies of atrial fibrillation and underscore the importance of flexibly selecting different sources for neutrophil isolation in various experimental animals with differing neutrophil distributions.

Protocol

The study was performed under a project license (No. 20211404A) granted by the Animal Ethics Committee of West China Hospital, Sichuan University, in compliance with the guidelines of the Animal Ethics Committee of West China Hospital, Sichuan University for the care and use of animals. In accordance with ethical guidelines, the rats used in this study were maintained in a controlled environment with a 12 h light/dark cycle, temperature at 22-24 °C and humidity of 50%-60%. The rats were given access to food and water ad libitum. The animals used in this study were 6-8 weeks old Sprague Dawley (SD) male rats, weighing about 250 g and specific pathogen-free. The animals were obtained from a commercial source (see Table of Materials).

1. Isolation of rat neutrophils

1. Bone marrow harvesting
   1. Place the rat in an appropriate container for anesthesia (see Table of Materials). Administer 3% isoflurane to the rat until the animal is unconscious. Check for the absence of a response to a toe pinch or tail pinch to ensure the depth of anesthesia.
   2. Once the rat is deeply anesthetized, perform cervical dislocation by placing the rat on its back and holding its tail with one hand while grasping the head with the other hand. Dislocate the neck quickly and firmly by applying a sudden and strong upward force on the tail while pulling the head downwards until the cervical spine is severed. Wait for confirmation of death, such as cessation of breathing and lack of heartbeat, before proceeding with tissue collection or disposal.
   3. Dip the rat in 75% ethanol and let it sit for a few minutes to sterilize the fur and skin. Lay the rat on its back and sever the lower limbs at the hip to protect the femur head. Use dissection scissors to cut through the ligament at the hock joint and fold the knee joint backward to expose the femur and tibia.
   4. Using forceps and scissors, carefully remove any muscles, tendons, and other tissues connected to the bones. Rinse the bones with Hank's Balanced Salt Solution (HBSS) to remove any remaining tissue debris and blood. Repeat two more times, for a total of three washes. Place the cleaned bones into a sterile container.
   5. Use a 5 or 10 mL syringe with a needle to poke the marrow through both ends of the bone to loosen the marrow matrix. Rinse the bone marrow with 10-15 mL Roswell Park Memorial Institute (RPMI) media with another syringe, until no visible bone marrow can be flushed out.
   6. Centrifuge the bone marrow cells at 300 x g for 10 min at 20 °C. Add 5-10 mL red blood cell lysis buffer (see Table of Materials) to resuspend the cells and incubate at room temperature for 3 min. Add 4 times the volume of HBSS to the mixture. Centrifuge the cells at 600 x g for 5 min at room temperature, discard the supernatant with a pipette, and use the resulting pellet for further use.
2. Isolation of neutrophils from bone marrow
   1. For the two-step method, perform the following additional steps:
      1. Isolate bone marrow cells using the commercialized rat neutrophil isolation kit following the manufacturer's instructions (see Table of Materials).
      2. Prepare a density gradient by layering 2 mL of 55% percoll reagent (see Table of Materials), 2 mL of 65% of the reagent, 2 mL of 70% reagent, and 2 mL of 80% reagent in a new 15 mL centrifuge tube. Centrifuge the tube at 800 x g for 40 min at 20 °C, with the acceleration set to 5 and the deceleration set to 0 or 1.
      3. Collect the 70% gradient layer, including the cell layers at the boundary between 65% and 70% gradient reagent, using a sterile pipette. Transfer the cell suspension to a new 15 mL centrifuge tube. Add 15 mL HBSS into the tube and gently invert the tube several times to wash the cells. Centrifuge the tube at 300 x g for 10 min at room temperature to pellet the cells.
   2. For the one-step method, perform the following additional steps:
      1. Subject bone marrow cells to the gradients without using the rat neutrophil isolation kit.
      2. Collect the 70% gradient layer, including the cell layers at the boundary between 65% and 70% gradient, using a sterile pipette. Transfer the cell suspension to a new 50 mL centrifuge tube and wash the cells with HBSS. Centrifuge the tube at 400 x g for 5 min at room temperature to pellet the cells.
      3. Count the isolated neutrophils using a hemocytometer and assess viability using trypan blue staining.
         ​NOTE: The protocol can be modified depending on the number of rats and the desired number of isolated neutrophils. An acceptable quantity of bones is obtained from three rats when this method is used for the first time in a new experimental setting. All procedures should be performed under sterile conditions. The isolation of neutrophils from bone marrow should be performed as quickly as possible to minimize cell damage and loss of viability.

2. Acquisition of rat NETs

1. Resuspend 0.5 x 10⁸-1 x 10⁸ isolated neutrophils in 4 mL RPMI media (supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin) in a sterile 10 cm Petri dish.
2. Add 500 nM PMA (see Table of Materials) to the neutrophil suspension to induce NETosis. Incubate for 3 h at 37 °C and 5% CO₂.
3. For the negative control, add DNase I (10 U/mL) into the neutrophil suspension to degrade the secreted NETs.
4. To harvest the NETs, remove the media and gently wash the NETs attached to the Petri dish with HBSS. Then, use intense flushing with 4 mL of fresh media for each plate to detach the NETs from the plate.
5. Collect the washing medium and pipette frequently for complete resuspension of NETs.
6. Centrifuge the suspension at 300 × g for 10 min at 20 °C to remove any floating cells.
7. Transfer the suspension containing the NETs to a sterile tube and store at −20 °C for further use within 2 weeks.
   ​NOTE: NETs are sensitive to degradation, so it is recommended to use freshly harvested material for best results.

3. Verification of the presence of NETs

1. Fix the cells (from step 2.4) on coverslips with 4% paraformaldehyde for 15 min and NETs (from step 2.7) for 30 min to 1 h.
2. Invert the coverslip onto an PBS/PBST droplet on a paraffin film-covered test tube stand and repeat the washing process thrice for 5 min each.
3. Permeabilize the cells with 0.5% Triton-X-100 for 10 min at room temperature and wash three times in PBS/PBST for 1 min each. Seal the cells with the 10% normal donkey serum (see Table of Materials) at room temperature for 1 hour.
4. Incubate the cells with the anti-rat neutrophil elastase antibody and anti-rat myeloperoxidase antibody (see Table of Materials) overnight at 4 °C. Then, wash the coverslip in PBS/PBST three times for 5 min each.
5. Incubate the cells with the secondary antibody A488-conjugated donkey anti-rabbit IgG (H + L), A594-conjugated donkey anti-mouse IgG (H + L), and A594-conjugated goat anti-Mouse IgG1 (see Table of Materials) at room temperature for 1 h in the dark. Then, wash the coverslip in PBS/PBST three times for 5 min each.
6. Stain cell nuclei and NET skeletons with 10 mg/mL DAPI. Then, wash the coverslip in PBS/PBST three times for 5 min each.
7. Place a drop of mounting medium on a glass slide and invert the coverslip with cells onto it. Let it dry for 1 h for microscopic analysis with immersion lenses; otherwise, it's ready for inspection.
   ​NOTE: Great care must be taken when manipulating NETs, even after fixation, as they are extremely fragile and can easily be lost during preparation.

4. Quantification of NETs

1. Prepare Tris-EDTA (TE) buffer and PicoGreen (dsDNA assay reagent) working solution according to the manufacturer's instructions (see Table of Materials).
2. Prepare standards by diluting stock DNA to concentrations of 1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 µg/mL.
   NOTE: To quantify the concentration of NETs, the dsDNA assay kit (see Table of Materials) was employed, adhering to the manufacturer's guidelines. Tris-EDTA (TE) buffer and dsDNA assay reagents were readied using a 19-fold volume of double-distilled water or a 199-fold volume of TE buffer, respectively. Subsequently, standard solutions (1 ng/mL, 10 ng/mL, 100 ng/mL, and 1 µg/mL) were prepared, and each 50 µL of the sample was combined with 450 µL of TE buffer.
3. Add 50 µL of each sample to 450 µL TE buffer.
4. Add 100 µL of standards or samples along with an equal volume of assay reagent working solution to each well in a 96-well plate, including standards and samples.
5. Incubate the plate at room temperature for 2-5 min, avoiding direct light.
6. Read the samples using a fluorescence microplate reader with an emission spectrum of around 530 nm and an absorbance spectrum of around 480 nm.
7. Calculate the concentration of NETs in each sample using the standard curve generated by the standards.

5. Analysis of NETs secretion by cell cytometry

1. Add nucleus stain (see Table of Materials) to the cells incubated in the 96-well plate to reach a final concentration of 10 ng/mL and incubate for 30 min.
2. Add cell-free DNA (cfDNA, see Table of Materials) stain to the cells to reach a final concentration of 300 nM and incubate for 10 min.
3. Mix the fluorescent dyes by gentle pipetting. Do not discard the supernatant or wash the pellet.
4. Load the sample plate into the cytometer instrument.
5. Set the parameters for focus and exposure time for the different channels: blue (ex: 377/50 nm, em: 470/22 nm), usually with the exposure of 30,000 ms, green (ex: 483/32 nm, eM: 536/40 nm) usually with the exposure of 5,000 ms.
6. Capture highly uniform images of the entire plate at different channels.
7. Analyze the nucleus stain counts for different groups. If they are similar, NETs secretion can be determined by the cfDNA stain count.

Results

The protocol outlined herein delineates two distinct methods, each characterized by improved purification or streamlined steps. Both methods yielded approximately 0.5 x 10⁸-1 x 10⁸ neutrophils per rat. Flow cytometry analysis, employing the annexin V-FITC/PI apoptosis detection kit, exhibited cell viability above 90%, comparable to mouse and human counterparts (Figure 1). While lymphocyte contamination seemed inevitable during neutrophil isolation from bone marrow, the ...

Discussion

The isolation of neutrophils constitutes a pivotal step in studying NETosis, where the selection of an appropriate isolation method is paramount for obtaining dependable results. An important factor to weigh is the occurrence of lymphocyte contamination during isolation. Addressing this challenge is particularly significant when isolating rat neutrophils from bone marrow. Despite the distinct density range of neutrophils (1.0814-1.0919, with a peak at 1.0919) compared to lymphocytes (1.0337-1.0765, with a peak at 1.0526)...

Materials

Name	Company	Catalog Number	Comments
A488-conjugated donkey antirabbit IgG(H + L)	Invitrogen, USA	A32790
A594-conjugated donkey anti-mouse IgG(H + L)	Invitrogen, USA	A32744
A594-conjugated goat anti-Mouse IgG1	Invitrogen, USA	A21125
Anti-rat myeloperoxidase	Abcam, England	ab134132
Anti-rat neutrophil elastase	Abcam, England	ab21595
Celigo Image Cytometer	Nexelom, USA	200-BFFL-5C
DNase I	Sigma, USA	10104159001
fetal bovine serum (FBS)	Gibco, USA	10099141C
Hank’s Balanced Salt Solution (HBSS)	Gibco, USA	C14175500BT
Hoechst	Thermofisher, USA	33342
Isoflurane	RWD, China	R510-22-10
Mowiol	Sigma, USA	81381
Normal Donkey Serum	Solarbio, China	SL050
Paraformaldehyde	biosharp, China	BL539A
Penicillin-streptomycin	Hyclone, USA	SV30010
Percoll	GE, USA	P8370-1L
Phorbol 12-myristate 13-acetate (PMA)	Sigma, USA	P1585
Picogreen dsDNA Assay Kit	Invitrogen, USA	P11496
Rat neutrophil isolation kit	Solarbio, China	P9200
Red blood cell lysis buffer	Solarbio, China	R1010
Roswell Park Memorial Institute (RPMI) media	Hyclone, USA	SH30809.01B
RWD Universal Animal Anesthesia Machine	RWD, China	R500
Sprague Dawley (SD) rats	Dashuo, China
SytoxGreen	Thermofisher, USA	S7020
Tris-EDTA (TE) buffer	Solarbio, China	T1120
Triton-X-100	Biofroxx, German	1139ML100