Flow Cytometric Detection of CD133 and pNF-κB

Detection of CD133 and phosphorylated NF-κB was performed by flow cytometry as described elsewhere15 (link) with minor modifications. Briefly, 0.5 × 10⁶ cells were plated in 2 ml of cell growth media in six-well plates and incubated overnight at 37°C and 5% CO₂. Healthy, untreated A549 cells/A549-CS were used to detect CD133. For NF-κB detection, A549-CS were treated with IOX-101 for 24 h in a regular cell incubator. Cells were stained for 20 min in the dark with anti-CD133 FITC or phospho-NF-κB p65 PE antibodies in intracellular fixation and permeabilization buffer reagent (Thermo Scientific). After a couple of washes with washing buffer, the cells were resuspended in PBS and 5,000 events were acquired in a Guava easyCyte™ flow cytometer. Cells were gated for CD133 or phosphorylated NF-κB-positive events and analyzed using ExpressPro software from Millipore.