Granzyme a fitc

Intracellular staining was used to measure the lytic proteins perforin, granzyme A and granzyme B contained within the secretory granules of NK cells [27 (link), 42 (link)]. Surface expression of CD57 was measured as a marker for NK cell maturation [43 (link)]. The PBMCs were incubated with mAbs for CD56-PE-Cy7, CD16-BV711, CD3-BV510 and CD57-PE-cyanin-based fluorescent dye (CF)594 for 25 min. The PBMCs were then permeabilised with BD fixation/permeabilisation solution for 20 min, washed in BD perm/wash buffer and then incubated with mAbs including perforin-APC (Miltenyi Biotec, Cologne, BG), granzyme A-FITC and granzyme B-V450 (BD Biosciences, San Diego, USA) for 30 min which was followed by flow cytometric analysis.

Intracellular staining was used to measure granzyme A and granzyme B as previously described [7 (link)]. NK cells and K562 cells were plated and placed in the incubator at 37 °C for 4 h with 5% CO₂ at E:T 25:1 in RPMI-1640 supplemented with 10% FBS. NK cells were incubated with Monensin (0.67 μl/ml) and Brefeldin A (1 μl/ml) (BD Bioscience, San Jose, CA, USA) to interfere with degranulation and intracellular cytokine transport from the endoplasmic reticulum. NK cells were stained with monoclonal antibodies for granzyme A FITC (0.5 μg/20 μl) and granzyme B BV450 (0.125 μg/5 μl) (BD Bioscience, San Jose, CA, USA) for 30 min at 4 °C. Both control and treated NK cells were measured using flow cytometric analysis recording 10,000 events (Additional file 1).

The anti-human mAbs have been used: CD3 PE (from Beckman Coulter), CD3 PErCP, CD11b PE, CD14 APC, CD56 PE, CD56 APC, CD73 PE, CD107a FITC, Granzyme A FITC, Perforin FITC, pJNK PE (from BD Biosciences), HLA-DR FITC (Dako), CD44 FITC, CD105 FITC, Granzyme B PE, Granzyme K Alexa647 (Immunotools), CD90 APC, COX2 FITC (Cayman chemicals). Each flow cytometric analysis was controlled with isotype-matched mAbs. All flow cytometry-based experiments were performed on FACS Calibur using Cell-Quest Pro Software. Offline data analysis was done on Summit 5.1 software.