Neutrophil conditioned media collection and inhibition.

1. Isolate neutrophils from 6–8-week-old female C57BL6/J mice.
   • 1 μg/ml of Granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech) loaded in 5 mg of mesoporous silica rods suspended in PBS was subcutaneously injected into the left flank of C57BL6/J mice
   • After 3 to 6 days, neutrophils are isolated from MSRs, peripheral blood, and spleen using a mouse neutrophil isolation kit (Miltenyi Biotec) 
2. Freshly isolated neutrophils are incubated with 100 nM of Phorbol 12-myristate 13-acetate (PMA) in Hanks’ balanced salt solution (HBSS) with 0.25% BSA for 2 hours for activation.  
3. Neutrophils were then thoroughly washed with HBSS and cultured at the density of 1 million cells/ml in RPMI supplemented with 1% heat-inactivated FBS and 1% P/S for 12 hours. 
4. The conditioned media, containing neutrophil-derived factors, was collected and used after centrifugation to remove dead cells and debris. 
5. Neutrophil-free RPMI supplemented with 1% heat-inactivated FBS and 1% P/S was incubated at 37°C for 12 hours and used as control media for NeutCM. 
6. To block functionality of specific factors in NeutCM, NeutCM was incubated with neutralizing antibodies [100 ng/ml of anti-Cxcl2 (Novus Biologicals), 100 ng/ml of anti-Ccl3 (Novus Biologicals), 100 ng/ml of anti-Lipocalin2 (Novus Biologicals), 1 µg/ml of anti Mmp-9 (Invitrogen)] and their IgG controls overnight at 4°C and then added to cell culture media.

This protocol is included in our published work (Seo et al., Sci Transl Med, 2021)