At present I am member of the Laboratory of Clinical Neuroimmunology in Institut de Recerca del Hospital Universitari Vall d'Hebron (Barcelona, Spain). We are concerned with Multiple Sclerosis research and now we are isolating Treg cells from human blood. I have read researchers to obtain about million cd4+cd25+cd127lo/- cells from 20 mL blood sample. After several attempts, we get an average of half a million cells from 64 mL blood sample. We use the CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II (MACS, from Miltenyi Biotec). We use either one LD column and two MS columns or two LD columns and one MS column. Could I know which protocol do you follow? Thanks.