Mnase reaction buffer

Frozen testis cryosections were thawed on a slide drier at 42 °C. Antigen retrieval was performed in boiling citrate buffer (pH 6.0) for 1 min, following which slides were washed twice in PBS for 5 min each. Next, testes sections were treated with 50 µl aliquots of MNase prepared at 2, 20, and 200 Kunitz units in MNase reaction buffer (New England BioLabs; M0247S) and incubated for 15 min at 37 °C. Controls (no MNase) were included and consisted of testis sections incubated with reaction buffer alone. MNase activity was stopped by adding 1/10th volume of 100 µM EDTA. Testis sections were washed in PBS before proceeding to immunofluorescence. MNase digestion was assessed by staining DNA with DAPI.

PMA-induced NETs were partially digested with 10 U/mL of Micrococcal nuclease (MNase; New England Biolabs [NEB]) in the presence of MNase reaction buffer (NEB) for 20 minutes at 37°C. The reaction was stopped by adding EDTA (MilliporeSigma) to a final concentration of 15 mM. NET protein concentration was determined using Bicinchoninic Acid Kit (Pierce) according to the manufacturer’s instructions.