Ao pi dye

HSC migration assays were performed using transwells. Briefly, 150,000 HSCs were suspended in 100μl of starvation media (IMDM containing 1% (vol/vol) BSA fract V) and was added to the upper chamber of the 5-μm-pore transwell insert (24-well plate format transwell, Corning, NY, USA). 0.5ml of starvation media with various concentrations (0–100ng/ml) of chemokine stromal derived factor-1 alpha (SDF-1α) was added to the bottom chamber. The transwell plates were incubated for 4 hours in a 37°C 5% CO₂. The transwell inserts were carefully removed and the migrated cells in the bottom chamber were resuspended. Samples were obtained from the bottom chamber and stained with acridine orange and propidium iodide nuclear dyes (AO-PI dye, Nexcelom, MA, USA). The stained samples were enumerated using Nexcelom auto 2000 cell counter (Nexcelom, MA, USA). All the migration experiments were performed in triplicates. In the experiments involving inhibitor treatments (LYN/Src inh. – RK20449 (Selleck Inc. TX, USA), SHIP1 inh. – 3AC (EMD Millipore Inc. MA, USA) and SHP1 inh. – TPI-1 (Cayman Inc. MI, USA)), the cells were exposed to inhibitors for 1 hour in IMDM containing 1% BSA media prior to adding to the top chamber of the transwell. Migration assays with TF1 cells were performed as described previously(27 ).