Trypan blue exclusion test

For the trypan blue exclusion test, 5 × 10⁴ cells per well were seeded in 12-well plates and cell growth was monitored every 24 h for 3 days, by counting the total number of viable cells per well. For this, cells were detached with trypsin and resuspended in 1 ml culture media. Next, 10 μl trypan blue (0.4%; Sigma-Aldrich) was added to 10 μl of cell suspension and 10 μl of this mix was loaded in a hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). Only viable (nonstained) cells were considered for the count.