An Assay to Monitor Degranulation of Azurophilic Granules in Neutrophils following Activation

Protocolo

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

1. Prepare mouse bone marrow neutrophils

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 phosphate-buffered saline (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 form 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 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 Roswell Park Memorial Institute 1640 (RPMI1640) media at 3 x 10⁶ cells/ml and kept on ice before the experiment. Cells are used 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% sodium chloride (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 underlay with 12 ml of 74% Percoll very gently.
   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 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 a concentration of 3 x 10⁶ neutrophils/ml 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-Formyl-Methionyl-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 supernatant, 100 μl, was examined for degranulation assay.
2. Measurement of degranulation of azurophilic granules
   1. Mouse neutrophils (2 × 10⁷ in 50 µl) in HBSS buffer containing 1 mM calcium chloride (CaCl₂) and 1 mM magnesium chloride (MgCl₂) were stimulated with or without 0.5 or 10 µM fMLP or 5 ng/ml TNF-α for 10 minutes at 37°C. 
   2. Cells were spun at 800 g for 5 minutes twice to collect 45 μl supernatant.
   3. The supernatant was run as duplicate samples.
   4. Incubate 20 μl supernatant with 80 μl 0.75 mM hydrogen peroxide (H₂O₂) solution and 100 μl 3,3',5,5'-tetramethylbenzidine Ready-to-Use solution at room temperature in the dark to detect the activity of myeloperoxidase (MPO). 
   5. After 30 minutes, the reaction was quenched with the addition of 0.8 N hydrochloric acid (HCl), and the absorbance was read on a PHERAstar plate reader at 450 nm.
   6. If necessary, recombinant MPO can be used to generate a standard curve to calculate the absolute amount of MPO in the sample.

Table 1. Component of Percoll gradient solution

	55% Percoll (30 ml)	74% Percoll (30 ml)
Percoll (ml)	16.5	22.2
9% NaCl (ml)	1.83	2.47
0.9% NaCl (ml)	11.67	5.33

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Materiais

Name	Company	Catalog Number	Comments
EnzChek Gelatinase/Collagenase Assay kit	Thermo Fisher Scientific	E12055	Used for specific granule degranulation assay
3,3',5,5'-tetramethylbenzidine Ready-to-Use solution	Thermo Fisher Scientific	J61325.EQE	Used for azurophilic granule degranulation assay
H2O2	Millipore Sigma	H1009	Used for azurophilic granule degranulation assay
Gibco™ RPMI1640	Thermo Fisher Scientific	21875034	Used for culturing neutrophils
BSA	Sigma	A3294	Used for culturing neutrophils
Percoll	Cytiva	17089101	Used for isolating neutrophils
HBSS	Thermo Fisher Scientific	14170112	Used for isolating and stimulating neutrophils
HEPES	Sigma	H4034	Used for isolating neutrophils
10x PBS	Thermo Fisher Scientific	70013032	Used for isolating neutrophils
Recombinant murine TNF-α	Peprotech	315-01A	Used for stimulating neutrophils
fMLP	Sigma	F3506	Used for stimulating neutrophils
PHERAstar FSX plate reader	BMG Labtech
Citrate-dextrose solution (ACD)	Sigma	C3821	Used for isolating human neutrophils
Dextran T500	Pharmacosmos	5510 0500 9007	Used for isolating human neutrophils