Direct immunofluorescence

A portion (200–300 μL) of the same samples prepared for direct flow cytometry (DFC) analysis, was concentrated by centrifugation at 2700 × g for 5 min and resuspended in PBS (20 μL) mixed with Fluoromount^™ (2 μL). After that, 10 μL of the cell suspension was pipetted on glass slides in duplicates and covered with coverslips. The slides were observed under confocal fluorescence microscopy (Nikon A1R MP, NIKON Instruments Inc., Japan) and the resulting images were analysed using Fiji software.