Human specimens and cells

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You can find a detailed protocol in our publication on how to isolate and generate NETs, Carmona-Rivera, C. and Kaplan, M.J. 2016. Induction and quantification of NETosis.Curr. Protoc. Immunol.115:14.41.1-14.41.14.doi: 10.1002/cpim.16.

1. Add 20 mL of Ficoll-paque to each of the two 50 mL conicals. SLOWLY layer blood from the green top tubes on top of the Ficoll. Divide the blood so that there is an equal total volume between the two conical tubes. Leave the red top tube as it is. 
2. Balance the tubes (red top and conicals) in a centrifuge. Spin at 1440 rpm, 20 min, zero accel /brake (takes 30min). 
3. To isolate monocytes, collect PBMC/buffy coat layer pipette from the top of the buffy to within 5 mL of RBC layer. Put it in an empty 50mL conical (note: one buffy coat per conical). Bring the volume up to 30 mL with PBS. Spin this down in a balanced centrifuge at 1600 rpm, 10 min, 9 = accel /brake. 
4. Take out ~8 mL of red blood layer. Mix this with ½ that volume (4 mL) of 20% Dextran. Shake and allow to sit for 5 min at room temperature. 
5. Then add PBS up to a volume of 30 mL. Shake and let sit for 30 min at room temperature.
6. Neutrophils will be in the top layer. Take off the top layer, bring up to 50 mL in PBS and spin down 1600 rpm for 5 minutes.
7. Resuspend pellet in 20 mL of 0.2%NaCl for 1min then quench with 30 mL of  1.8% NaCl.
8. Centrifuge at 1,600 rpm. And resuspend pellet in PBS.  Count cells and resuspend neutrophils in RPMI at a desired density.
9. Set up a 24-well plate with 10 million neutrophils/mL. Treat them with 1 uM of PMA
10.  Incubate plate for 4 h at 37C to generate NETs.
11. harvested supernatant and digest NETs with 10 U/mL micrococcal nuclease (Thermo) for 15 min at 37°C. 
12. Collect Supernatants and spin at 5,000 rpm for 5min to remove intact cells. Transfer supernatant into a fresh tube ( these are your harvested NETs), store at -20 or -80C,
13. Monocytes from PBMC layer were isolated according to manufacture’s recommendations, 130-050-201For research use only  CD14 MicroBeads, human
14. Monocytes were transformed into osteoclasts by stimulation with 50 ng/mL of M-CSF for 3 days followed by 50 ng/mL RANKL.