I tried to do proliferation assay with PBMC and MSC. I have followed cells proliferation with CFSE. I activate PBMC with PHA-M(2,5microg/ML) and I labeled PBMC with 1microM CFSE But after 72 h, it seems so much cells under microscope but when I collect them and wash them with PBS and count them they are not so much and they are less than day 0?! It seems that PBMC  attached to my plate or my tube during washing and culturing?! I used 6 well flat cell culture plate. moreover I washed PBMC with cold PBS two times but it doesn't help !. Has anyone some good tips?

Thanks in advance.

Best regards

//Bahareh  Khalaj