MUB40 is a novel neutrophil marker, which binds to lactoferrin and allows a specific detection in human and all mammal tissue sample tested so far. Labeling neutrophils with MUB40 is easy, quick, and specific. It's now used widely in our lab and the implication of this technique extend towards diagnosis of inflammatory diseases.
To begin, prepare solutions one, two, and three according to the text protocol, and place them in an anoxic cabinet overnight. Then, centrifuge the tubes of collected blood at 650 g for 20 minutes to separate the cells from the plasma fraction. Without disturbing the separated blood, transfer the tubes to the anoxic cabinet and transfer the plasma fractions to a 50 milliliter conical tube.
After this remove the tube of plasma from the anoxic cabinet and centrifuge it at 2900 g for 20 minutes. Taking care not to disrupt the pelleted platelets, transfer the tube to the anoxic cabinet and pipette the platelet poor plasma into a fresh 50 milliliter tube. Using the blood collection tubes, combine the red blood cells in a conical 50 milliliter tube.
Then, add 0.9%sodium chloride solution until the total volume reaches 44 milliliters, and add six milliliters of 6%dextran. Invert the tube 10 to 20 times to gently mix the blood and dextran mixture, and allow the tube to sediment for at least 30 minutes. While the tube sediments, prepare a Percoll gradient by adding 4.2 milliliters of Percoll solution to 5.8 milliliters of plasma, and invert the tube to mix.
Next, collect the neutrophil containing top fraction of the dextran, taking care not to pipette red blood cells. Tighten the screw cap, remove the dextran tube from the anoxic cabinet, and centrifuge the tube at 300 g for 10 minutes. Taking care not to disturbed the pelleted cells, place the tube back in the anoxic cabinet.
Then, remove the liquid by pipetting. Gently resuspend the pellet in one milliliter of plasma. Slowly add the resuspended cells to the tops of the Percoll solution.
Carefully remove the Percoll solution tube from the anoxic cabinet and centrifuge it at 800 g for 20 minutes to separate PBMCs from neutrophils. Next, prepare 14 milliliters of washing buffer solution according to the text protocol. Then, place the Percoll tube in the anoxic cabinet.
Use a pipette to remove the Percoll and PBMCs and resuspend the neutrophil containing pellet in one milliliter of washing buffer solution. Next, add 200 microliters of magnetic beads to the resuspended cells. Gently mix and incubate the cells and beads for at least 15 minutes.
After this, wash a separation column twice, with two milliliters of washing buffer. While holding a new, 15 milliliter conical tube under the column, slowly pipette the neutrophil red blood cell mixture through the column. The flow-through from the column should be cloudy and lose its red color, confirming the total separation of neutrophils from remaining red blood cells.
Add two milliliters of washing buffer to the top of the column and collect the flow-through in the same tube. By the end of this wash, the flow-through should be clear. Gently mix the collection tube to ensure the neutrophils are evenly distributed.
Then collected 10 microliters of the flow-through from cell counting. After this, remove the neutrophil tube from the anoxic cabinet and centrifuge the tube at 300 g for 20 minutes. Place the sedimented neutrophils back into the anoxic cabinet and remove the washing buffer via pipetting.
Then, resuspend the pellet in plasma. Add one microliter of RI-MUB40-Cy5 to one milliliter of RPMI-1640 medium, without phenol red, and mix via pipetting. Then, add one microliter of FMLP solution and pipette to mix.
Transfer the mixture to a glass bottom microscopy dish. Then add an appropriate volume of purified cells to the dish. Finally, place the dish into an inverted fluorescent microscope and start image acquisition.
In this protocol, MUB40 was used on living cells to detect neutrophil secretion upon simulation with FMLP. The dynamics of the release of granule content, including lactoferrin, may be visualized over time, here in magenta. MUB40 may be additionally used on fixed neutrophils.
Here, phagocytizing fluorescent beads in a timed series. Neutrophil granule's content is labeled here in blue. The neutrophils showed little to no cell staining at early time points and gradually, RI-MUB40 labeling increased over time, both on the plasma membrane and bead surface.
MUB40 may also be used on fixed inflammatory tissues to detect neutrophils specifically, as shown in the text figures. We describe here the neutrophil purification procedure, applied routinely in our lab. Any other method is suitable, MUB40-Cy5 labeling will still work.
Neutrophil may be specifically labeled with MUB40 after purification, or directly in inflamed tissue from patients or animal models. Several MUB40 derivatives have been designed to broaden its use. After its development, this technique paved the way for researcher in the field of inflammation, to explore neutrophilic recruitment and activation in inflammatory diseases.
