Separation and Optimisation of a Sucrose Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells (PBMC)
Sudeep Nagaraj *
Sapthagiri Institute of Medical Sciences and Research Centre, Bengaluru, India.
Shubha Nivargi
Sapthagiri Institute of Medical Sciences and Research Centre, Bengaluru, India.
Leelavathy Nanjappa
Sapthagiri Institute of Medical Sciences and Research Centre, Bengaluru, India.
Jagadish Tavarekere Venkataravanappa
Sri Devaraj Urs Academy of Higher Education and Research, Kolar, India.
*Author to whom correspondence should be addressed.
Abstract
One step centrifugation procedure used commonly for separation of blood cells is the ficoll gradient centrifugation. In this method, after centrifugation, the peripheral blood mononuclear cells (PBMCs) are located on the top of the separation fluid, whereas other blood cells erythrocytes and granulocytes sediment to the bottom. In the present study 75% of lymphocyte suspension could be separated by using a one-step density gradient centrifugation of sodium heparin blood with Sucrose. Sucrose was diluted into different concentrations using miliQ water (10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%,). 4 mL of diluted blood was layered on 4 mL of each sucrose solution and centrifuged for 45 minutes at 1000 rpm. Clear separation of PBMCs could be observed in solution with 40% sucrose. The separated PBMCs were analysed in haeme analyser which showed 75% lymphocytes, 23% monocytes and 2% of other cells.
Keywords: Sucrose, PBMCs, gradient centrifugation, blood.