Rapid Magnetic-microbead Method for Efficient Purification of Low-density Neutrophils

We study low-density neutrophils within peripheral blood mononuclear cells to characterize their functions in order to define their role in different diseases. Low-density neutrophils are rare in healthy blood, but they markedly increase in diseases such as systemic lupus erythematosus and cancer. To begin, add 10 units per milliliter of heparin as an anticoagulant into a 15-milliliter conical centrifuge tube.

Then add two milliliters of 6%dextran T500 in PBS. Obtain 10 milliliters of blood from a healthy adult volunteer by venipuncture. In another fresh 15-milliliter centrifuge tube, add five milliliters of density gradient medium.

Carefully pipette out the plasma without touching the erythrocytes and layer it on top of the medium to form two separate phases. Centrifuge the tube at 516 g for 20 minutes at four degrees Celsius. To isolate PBMCs, aspirate and discard the plasma above the PBMC band without disturbing the cells.

Collect the mononuclear cell band between the plasma and the medium, minimizing collection of the medium. Add 20 milliliters of PBS to the tube containing PBMCs, then centrifuge at 400 g for five minutes at four degrees Celsius. Carefully aspirate out the supernatant and scrape the tube to separate the pellet.

Add 10 milliliters of cold PBS to resuspend the cells. To isolate neutrophils, scrape the tube to separate the cells after pipetting out the medium. Then add 10 milliliters of cold PBS.

Now transfer the cells into a fresh 50-milliliter conical centrifuge tube and centrifuge. Aspirate the supernatant and scrape the tube again. Now pipette 10 milliliters of cold hypotonic solution into the tube and mix gently.

Mix gently for exactly one minute. Quickly add 10 milliliters of cold hypertonic solution to make the solution isotonic. Then count the neutrophils using a Neubauer chamber and ensure the purity is greater than 95%Centrifuge the suspension to obtain a cell pellet.

Then resuspend the pellet in cold PBS. Centrifuge the peripheral blood mononuclear cells at 400 g for five minutes at four degrees Celsius. After removing the supernatant, resuspend the cells in 120 microliters of cold wash buffer.

Then pipette 35 microliters of CD66b magnetic microbeads to the cell suspension. Incubate the mixture in the dark at four degrees Celsius for 30 minutes. Now pipette one milliliter of cold wash buffer to the tube.

Centrifuge the tube at 400 g for three minutes. Scrape the tube to separate the cell pellet after removing the supernatant. And resuspending the cells in one milliliter of wash buffer.

Place a magnetic separation column onto a magnet. Add 0.5 milliliters of wash buffer to the column, allowing it to pass through completely. Transfer one milliliter of the resuspended cells onto the column and let the buffer pass through drop by drop.

After washing, transfer the column into a microcentrifuge tube. Then pipette one milliliter of wash buffer into the column. Now insert the plunger on top of the column.

Gently apply pressure to elute the cells. Then remove the plunger and place the column onto a new microcentrifuge tube. Add another milliliter of wash buffer to the column.

Then insert the plunger again and gently apply pressure to elute the remaining cells. Centrifuge both microcentrifuge tubes at 800 g for three minutes. Resuspend the cell pellets from both tubes into one milliliter of cold PBS.

Keep the cell suspension on ice. Resuspend the purified cells in labeling buffer made of 1%fetal bovine serum in PBS. Transfer 250 microliters of the suspension into a 1.5-milliliter microcentrifuge tube.

Add the corresponding antibodies against neutrophil membrane molecules to the tube. Then incubate the cells for 30 minutes at four degrees Celsius, protected from light. Now pipette one milliliter of PBS to the tube.

Spin the tube in a microcentrifuge at 800 g for three minutes. Aspirate out the supernatant. Tap the tube to break the pellet.

And resuspend the cells in 0.5 milliliters of 1%paraformaldehyde. Then keep the cells at four degrees Celsius, protected from light until analysis by flow cytometry. Analyze the samples using flow cytometry, capturing 10, 000 events per sample.

Low-density neutrophils in healthy individuals represented approximately 5%of the peripheral blood mononuclear cells, while the described magnetic isolation protocol yielded low-density neutrophils at about 98%recovery. Neutrophils express the membrane markers CD10, CD11b, CD15, CD62L, and CD66b. Magnetically purified low-density neutrophils expressed the same membrane markers as neutrophils.

Purified low-density neutrophils exhibited a multilobulated nucleus and were similar in size to neutrophils. Both neutrophils and low-density neutrophils generated reactive oxygen species in response to PMA stimulation, with low-density neutrophils producing higher levels than neutrophils. Low-density neutrophils released neutrophil extracellular traps in response to PMA stimulation as evidenced by colocalization of DNA with elastase and with citrulline.

Both purified neutrophils and low-density neutrophils released NETs with similar kinetics and amounts after PMA treatment. Our protocol provides a fast and reproducible way to obtain highly pure and functional low-density neutrophils. We address the lack of a standardized, time-efficient protocol for isolation of low-density neutrophils from human blood.

This protocol does not require any special training for complex instruments, and it's also more economical and faster than FACS.