Samir Ranjan Panda thanks in advance if you may advise on this.

Hi Dina Ahmed

These are multiple layered cells growing on top of each other mainly because of reduced seeding density and clumps formation which causes only specific population of cells to grow. Mix thoroughly after trypsinization to prevent clumps and layered growth otherwise the cells look fine in Fig-1.

Thanks,

Samir Ranjan Panda Thank you so much for the valuable answer. May you kindly confirm that this is due to the high or low seeding density? I did it in 24 well plate at 0.5 x 10^6 (half a million) per well.

thank you so much.