Il 6 fitc

For cytokine detection in irradiated MRC5 cells, secretion of cytokines was blocked for 18 hours with a protein transport inhibitor Brefeldin A (eBioscience). Staining of intracellular cytokines was performed according to manufacturer's protocol. Briefly, after the incubation with Brefeldin A, cells were harvested by trypsinization, washed once in PBS and fixed with IC Fixation Buffer. Cells were incubated in the dark at room temperature for 20 min. Then, 1x Permeabilization buffer was added directly to cells with fixation solution. Samples were centrifuged at 400 x g for 5 min at room temperature. The cell pellet was re-suspended in 100 μl of 1x Permeabilization buffer containing fluorochromes-labelled antibody for detection of intracellular cytokines and incubated in the dark at room temperature for 20 min. Following antibodies were used for staining: IL-1α-FITC (eBioscience), IL-1β-FITC (eBioscience), and IL-6-FITC (eBioscience). After the incubation period, 1x Permeabilization buffer was added and samples were centrifuged at 400 x g for 5 min at room temperature. The cell pellet was then re-suspended in Flow Cytometry Staining buffer and samples were centrifuged again at 400 x g for 5 min at room temperature. Stained cells were then re-suspended in Flow Cytometry Staining buffer and were processed for cytokines expression analysis by flow cytometry.