Answer No.1 : Lymphocyte Separation Medium (LSM) is a sterile, iso-osmotic polysucrose and diatrizoate solution with low viscosity originally designed for the in vitro isolation of lymphocytes from diluted whole blood. Differential migration following centrifugation will result in the formation of several cell layers. Mononuclear cells (lymphocytes and monocytes) and platelets will be contained in the banded plasma-LSM interphase due to their density. The pellet that is formed contains mostly erythrocytes and granulocytes, which have migrated through the gradient to the bottom of the tube. Lymphocytes are recovered by aspirating the plasma layer and then removing the cells. Excess platelets, LSM, and plasma can then be removed by cell washing.

Method of separatio is as follows:

1. Thoroughly mix the LSM by inverting the bottle gently.
2. Aseptically transfer 3 mL of LSM to a 15 mL centrifuge tube.
3. Mix 2 mL of defibrinated or hepranized blood with 2 mL of physiological saline or balanced salt solution.
4. Carefully layer the diluted blood over 3 mL of LSM (room temperature) in a 15 mL centrifuge, creating a sharp blood-LSM interphase. DO NOT MIX! The quality of the separation is dependent upon a sharp interphase between the lymphocytes and the solution.
5. Centrifuge the tube at 400 x g at room temperature for 15 to 30 minutes. Centrifugation should sediment erythrocytes and polynuclear leukocytes and band mononuclear lymphocytes above the LSM.
6. Aspirate the top layer of clear plasma to within 2 - 3 mm above the lymphocyte layer.
7. Aspirate the lymphocyte layer plus about half of the LSM layer below it and transfer it to a centrifuge tube. Add an equal volume of buffered balanced salt solution to the lymphocyte layer in the centrifuge tube and centrifuge for 10 minutes at room temperature (18 to 25°C) at a speed sufficient to sediment the cells without damage, i.e., 160 - 260 x g. Washing the cells removes LSM and reduces the percentage of platelets.
8. Wash the cells again with buffered balanced salt solution and resuspend in the appropriate medium for your applications.

Answer No.2: B & T Lymphocytes can be differentiated by their CD markers on the surface. For example, T Lymphocyte can be identified by presence of CD3 whereas B lymphocyte by the presence of CD19 & CD20. T lymphocyte can be either T helper or T cytotoxic. In this case both will have CD3, however T-helper has CD4 and T cytotoxic has CD8. Flow cytometry technique can be used to detect surface CD marker.

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