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Neutrophils are a type of white blood cell. As the most abundant cell in the innate immune system, they participate in natural immunity and regulate adaptive immunity. They are the most numerous immune cells in the blood and have phagocytic function. They play a key role in the body's anti-infection process.

Compared with the peritoneal cavity and blood, the mouse bone marrow is a huge neutrophil reservoir. Under steady-state conditions, there are a large number of immature and mature neutrophils in the bone marrow, which far exceeds the neutrophil reserves in the blood and peritoneal cavity.

Therefore, we will share the experimental method of isolating and extracting neutrophils from mouse bone marrow. It provides an ideal sample source for studying their biological characteristics, functional mechanisms, and roles in immune responses. This method is designed to help researchers extract neutrophils efficiently and purely, providing support for subsequent cell function analysis, signaling pathway research, and drug screening experiments.

Materials and instruments required

Biological materials

Experimental animals: C57BL/6 mice

Reagents

Solutions and culture media:

70% (v/v) ethanol solution

RPMI 1640 containing glutamine and HEPES

FBS (fetal bovine serum)

Penicillin/streptomycin mixed solution

PBS (phosphate buffered saline)

0.2% and 1.6% sodium chloride (NaCl) solution

PBS containing EDTA

Staining and antibody reagents:

LIVE/DEAD fixable blue dead cell staining kit

Anti-mouse CD45 APC antibody

Anti-mouse Ly6G PE antibody

Anti-mouse CD16/CD32 blocking antibody

Enzymes and reagents:

DNase I

Percoll (density gradient centrifugation separation reagent)

Casein solution

Anesthetic and anticoagulant reagents:

Methoxyflurane (for mouse anesthesia)

Heparin (for anticoagulation)

Instruments and consumables

Centrifugal instruments and consumables: Welso desktop low-speed refrigerated centrifuge, 15mL or 50mL centrifuge tubes

Sterile operation tools: sterile scissors, sterile tweezers

Cell processing tools: 70 μm cell strainer

Pipette tools: 3 mL, 10 mL and 25 mL serological pipettes

Culture consumables: cell culture plates or cell culture dishes (select specific specifications according to experimental requirements)

Others: alcohol lamp or other sterilization device to maintain a sterile operating environment

Experimental procedures

Mouse euthanasia

Control mice were euthanized according to approved IACUC procedures and the animal surfaces were disinfected by spraying with 70% ethanol.

Expose the abdomen and lower limbs

Use scissors to make an incision in the middle of the mouse abdomen, remove the skin, and expose the abdomen and lower limbs.

Removing muscle and dislocating the hip joint

Using scissors, remove the muscle from both legs and carefully dislocate the acetabulum from the hip joint.

Prepare bones

Perform subsequent steps under a tissue culture hood to prevent contamination and activation of neutrophils. Place the bones in a 100 × 15 mm sterile culture dish containing 15 mL of pre-chilled 1x RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution and place the dish on ice.

Removal of remaining muscles and separation of femur and tibia

Use a scalpel to further remove the remaining muscles of the femur and tibia, and separate the femur from the tibia at the knee joint.

Rinse the bone surface

Rinse each bone with 70% ethanol solution and then wash three times with 15 mL of ice-cold sterile PBS in the corresponding Petri dish to remove the ethanol from the bone surface.

Removal of bone marrow

Use a scalpel to remove the epiphysis of the bone marrow cavity and place it in a sterile empty culture dish.

Using a 25G × 5/8 needle and a 12 mL syringe with 10% FBS RPMI 1640 medium and 2 mM EDTA, flush bone marrow cells through a 100 μm cell strainer into a 50 mL conical tube.

Cut the bone marrow and filter

Use a scalpel to cut the bone marrow cavity into small pieces of 0.5–1 mm³ and pass them through a 100 μm cell strainer into a 2.5 mL EP tube.

Bone marrow cells were collected by centrifugation

Bone marrow cells were collected by centrifugation at 427 g for 7 minutes at 4°C.

Lysis of red blood cells

Using a 25 mL serological pipette, resuspend the cell pellet in 20 mL of 0.2% NaCl solution for approximately 20 seconds, then add 20 mL of 1.6% NaCl solution to lyse the red blood cells.

Centrifuge and collect cells

Centrifuge at 427g for 7 minutes at 4°C to collect bone marrow cells.

Wash cells

Use a 10 mL serological pipette to resuspend the cell pellet in 1x RPMI 1640 medium containing 10% fetal bovine serum, add 2 mM EDTA, wash the cells again, and repeat the previous centrifugation step.

Count live cells

Count live cells using trypan blue staining and resuspend the cells in 1-3 mL of chilled sterile PBS.

Prepare Histopaque layer

Add 3 mL of Histopaque 1119 (density 1.119 g/mL) to a 15 mL conical centrifuge tube and carefully layer 3 mL of Histopaque 1077 (density 1.077 g/mL) on top.

Loading cell suspension

Carefully overlay the bone marrow cell suspension on the Histopaque 1077 layer.

Centrifugation and stratification

Centrifuge at 872g for 30 minutes at room temperature to form a density gradient.

Collect neutrophils

Use a 3 mL pipette to collect neutrophils at the interface of the Histopaque 1119 and Histopaque 1077 layers.

Washing neutrophils

Collect neutrophils in 1x RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin and centrifuge at 427g for 7 minutes at 4°C.

Determination of cell purity and viability

A small aliquot was taken before the last wash to determine cell viability using the trypan blue exclusion method; the cells were also stained with fluorescent viability dyes and analyzed for cell purity by flow cytometry (FACS). Neutrophils were labeled with APC-conjugated anti-CD45 antibody, PE-conjugated anti-Ly6G antibody, and APC-Cy7-conjugated anti-AD11b antibody to accurately determine purity.

Through the above steps, high-quality bone marrow cells can be effectively extracted from the mouse bone marrow, providing necessary cell samples for subsequent immunological analysis, cell function experiments, etc.

Precautions

Instrument sterilization

Before the experiment, ensure that all instruments are sterilized by high pressure, and liquid reagents are sterile filtered to ensure aseptic operation.

Bone marrow flushing

When using saline to flush the bone marrow cavity, ensure that the bone marrow cavity turns white and that the bone marrow cells are completely released.

Aseptic operation

To ensure the success of subsequent cell culture, the entire operation process must be strictly aseptic to avoid any contamination.

Histopaque layering operation

The volume ratio of saline cell resuspension to Histopaque 1077 is 1:3. When adding, it should be added slowly along the wall of the tube to avoid mixing.

The volume ratio of saline cell resuspension to Histopaque 1119 is 1:2. When adding, it also needs to be added slowly along the wall of the tube to ensure clear stratification.

Collection of layered interface

In the density gradient after centrifugation, neutrophils are located in the cloudy middle layer of liquid. When collecting, the volume ratio of the aspirated liquid to the volume of saline added is 1:5 to ensure high-purity neutrophils.

From the above steps, we know that the desktop low-speed centrifuge is an indispensable key equipment in this experiment. Through reasonable density gradient centrifugation and cell resuspension operations, the target cells can be efficiently separated and the vitality and functionality of the cells can be effectively maintained. In actual operation, the specific centrifugation parameter settings need to be flexibly adjusted according to the experimental conditions and cell types to ensure the best separation effect. Optimizing centrifugation conditions, such as speed, time and temperature settings, will directly affect the cell purity and the reliability of the experimental results. Therefore, precise centrifugation operation is a key factor to ensure the success of the experiment.