A Degranulation Assay to Monitor Neutrophil Activation and Gelatinase Release

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Prepare mouse bone marrow neutrophil

1. Harvest bone marrow cells
   1. Bone marrow cells are flushed out from the femur and tibia of C57BL/6 mice with 10 ml Hank's balanced salt solution (HBSS) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.1% bovine serum albumin (BSA), pH 7.4 using a 10 ml syringe with 27 1/2G needle.
   2. Cells are filtered through 40 μM nylon meshes and centrifuged at 300 g at 4°C for 10 minutes.
2. Prepare 63% Percoll solution
   1. Prepare 100% Percoll solution by mixing 5 ml sterile 10x PBS with 45 ml Percoll (Cytiva).
   2. Mix 3.15 ml 100% Percoll solution with 1.85 ml HBSS in a 15 ml tube to make a 63% Percoll solution.
3. Isolate neutrophils from bone marrow cells
   1. Bone marrow cell pellets are resuspended in 1 ml HBSS; cells are laid carefully on the top of a 63% Percoll solution and centrifuged at 1,200 g at 4°C for 30 minutes without a break. Neutrophils are collected at the pellet.
   2. Red blood cells are lysed by a lysis buffer (Sigma), and cells are centrifuged at 800 g at 4°C for 5 minutes.
   3. Neutrophils are resuspended with RPMI1640 (Roswell Park Memorial Institute) media at 3 x 10⁶ cells/ml and kept on ice before the experiment. Cells are used up within 4 hours after isolation.

2. Isolation of human neutrophils

1. Collect leukocyte-rich layer from human blood
   1. Collect blood into a syringe filled with 10% volume of sterile acid citrate dextran (ACD).
   2. Pour blood into a 50 ml tube and add the same volume of 3% Dextran T500 in 0.9% NaCl.
   3. Let the mixture stay at room temperature for 10-15 minutes until red blood cells (the bottom layer) are separated from the leukocyte-rich layer (the top layer).
   4. Collect the top layer into a new tube.
2. Percoll gradient centrifuge
   1. Prepare 55% and 74% Percoll solutions, as mentioned in Table 1.
   2. Prepare the Percoll gradient in 50 ml tubes by adding 12 ml of 55% Percoll and gently underlay with 12 ml of 74% Percoll.
   3. Add the leukocyte-rich layer to the top of the Percoll gradient very gently.
   4. Spin cells at 800 g for 60 minutes at 12 ºC without a break.
   5. Remove the top mononuclear cell layer and harvest the neutrophil layer (around 5 -8 ml) into a new 50 ml tube containing 40 ml HBSS.
   6. Centrifuge at 800 g for 10 minutes at 4 ºC to pellet neutrophils.
   7. Resuspend the cell pellet with 1 ml red blood cell (RBC) lysis buffer and incubate it for 2 minutes at room temperature.
   8. Transfer the cell suspension into a 1.5 ml tube and add 500 µl HBSS.
   9. Centrifuge it at 800 g for 5 minutes at 4 ºC.
   10. Resuspend the cell at 3 x 10⁶ neutrophils/ml concentration in RPMI1640 media containing 0.5% BSA. Store isolated neutrophils on ice.

3. Specific granule degranulation assay

1. Neutrophil stimulation
   1. Neutrophils (2 × 10⁶/100 µl, 200 μl) in RPMI1640 were stimulated with or without 0.5 or 10 μM N-Formylmethionyl-leucyl-phenylalanine (fMLP) or 5 ng/ml tumor necrosis factor alpha (TNF-α) for 10 minutes at 37°C.
   2. Cells were then centrifuged at 800 g for 5 minutes.
   3. The supernatant was collected and spun for another 5 minutes at 800 g.
   4. The final 100 μl supernatant was examined for degranulation assay.
2. Measurement of degranulation of gelatinase granules
   1. The EnzChek Gelatinase/Collagenase Assay kit (Thermo Fisher Scientific) was used to measure the specific granule degranulation.
   2. According to the manufacturer's instructions, add 1 ml Milli-Q water to the lyophilized dye-quenched (DQ^TM) gelatin vial to make 1 mg/ml DQ^TM gelatin stock solution.
   3. Mix 18 ml Milli-Q water and 2 ml 10x reaction buffer into a 15 ml tube to make the reaction buffer.
   4. Add 0.5 ml Milli-Q water to the Clostridium collagenase tube to prepare a 1,000 U/ml stock solution. Before the experiment begins, dilute the collagenase standard solution using the reaction buffer to make the concentrations of collagenase 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, and 2 U/ml for generating a standard curve.
   5. In a 96-well plate, add 80 μl of the reaction buffer to each well.
   6. Dilute DQ^TM gelatin solution 4 times from stock solution with 1x reaction buffer before use. Then, add 20 μl of diluted DQ^TM gelatin solution to the wells of a 96-well plate to make the final concentration of DQ^TM gelatin 25 μg/ml in the reaction.
   7. Add 100 μl sample or different Clostridium collagenase standard solution concentrations into each well.
   8. Insert the assay plate into the PHERAstar plate reader (BMG Labtech) with an excitation wavelength of 485 nm and emission wavelength of 520 nm. The data point was collected every 10 minutes for 4 hours. Data were presented as a total activity using known concentrations of collagenase as a standard curve.

Table 1. Component of Percoll gradient solution.