Sodium azide

The susceptibility to collagenase digestion was studied in Collagenase A (from Clostridium histolyticum) solution. Approximately, 4 mg (dry weight in triplicates) of each sample were weighed. To the weighed samples, 0.2 ml aliquots of 2.5 mg/ml Collagenase A solution in Tris buffer (50 mM, pH 7.4) containing 10 mM calcium chloride and 0.02 mg/ml sodium azide (Paneco, Russia) were added. The samples were incubated at 37°C for 24 h. Then, the samples were centrifuged at 12,000 rpm for 10 min (using an A-14, Thermo Electron Corporation, Jouan-type centrifuge), washed with phosphate buffered saline (PBS), and the remaining tissue samples then dried in an oven at 60°C for 20 h. Finally, they were weighed and the weight loss was calculated by a paired comparison of before and after the treatments. The biodegradation in papain was studied in a solution of papain (Karipazim, Georgia, 100 U/ml) prepared in Tris buffer (50 mM, pH 8) containing 10 mM cysteine, 20 mM ethylenediaminetetraacetic acid (EDTA) and 0.02% sodium azide (Paneco, Russia) after 6 days, followed by the same measurement protocol.

The susceptibility to proteolytic degradation was studied in a Collagenase A (from C histolyticum) solution. Approximately 4 mg (dry weight in triplicates) of the sample were weighed. To the weighed samples, 0.5 mL aliquots of a 2.5 mg/mL Collagenase A solution in the Tris buffer (50 mmol/L, pH 7.5) containing 10 mmol/L calcium chloride and 0.02 mg/mL sodium azide (Paneco, Moscow, Russia) were added. The samples were incubated at 37 °C for 6 h. Then, the samples were centrifuged at 605 g (3000 RPM) for 90 s (a MiniSpin microcentrifuge by Eppendorf Corporation, Hamburg, Germany). We used a low rotation speed and a short time of centrifugation in order to better preserve the structure integrity for the following histological analysis. Then, the material was washed from the residual collagenase with distilled water. The precipitate was carefully transferred using a micropipette to a coverslip for the following drying in an oven at 50 °C for 20 h. Then, the dry residue was weighed using a WXTE ultramicrobalance (Mettler Toledo GmbH Urdorf, Switzerland). Finally, the weight loss was calculated by a paired comparison before and after the treatment.

The following mouse anti-human fluorescent-labeled antibodies were used for surface cell staining: CD56-APC-Vio770 (clone REA196), CD57-VioBlue (clone TB03), CD107a-APC (clone REA792), HLA-DR-PE-Vio770 (clone REA805), IFNγ-PE (clone 45-15), KIR2DL1-APC (clone REA284), KIR2DL2/DL3-PE (clone DX27), NKG2C-VioBright (clone REA205), HLA-E-PE (clone REA1031) (Miltenyi Biotec, Bergisch Gladbach, Germany); CD56-BrilliantViolet 421 (clone 5.1 H11), CD57-PE (clone HNK-1), CD57-APC (clone HNK-1) (Sony Biotechnology, San Jose, CA, USA); NKG2C-PE (clone 134591) (R&D Systems, Minneapolis, MN, USA).
PBMC/NK cells were washed in the PBA staining buffer (PBS containing 0.5% BSA (bovine serum albumin) (PanEco, Moscow, Russia) and 0.01% sodium azide (PanEco, Moscow, Russia) and incubated with antibodies for 30 min on ice in a PBA then washed twice in the same buffer before measurement. Flow cytometry analysis was carried out on a MACSQuant 10 cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) equipped with 405 nm, 488 nm, and 635 nm lasers.