The Lymphocyte Separation Medium LSM 1077 is a separation solution made with Ficoll^TM density gradient media. Ficoll^TM is a hydrophilic polymer with a molecular weight of 400.000 Dalton. It is used for the production of density gradients for the separation of cells and subcellular components, which sediment during centrifugation due to gravity.

According to the blood volume to be separated, either 15 or 25 ml size centrifuge tubes are filled with either 7 or 10 ml Lymphocyte Separation Medium (D= 1,077 g/ml, at +20°C). Heparinized whole blood is then, either undiluted or diluted with equal parts of PBS or culture medium, carefully poured over the lymphocyte separation solution. The separation process is performed by centrifugation at 1.200x g for 20 minutes.

The lymphocytes (70-100% enrichment) concentrate in the interphase (white layer) between the plasma and the separation solution. They are subsequently extracted with a sterile Pasteur pipette and washed twice in culture medium: 

1. Wash and spin for 10 min at 300x g
2. Wash and spin for 10 min at 200x g
3. The following cell counting is done by using the standard methods.

Preparation of solutions with other densities can be made by iso-osmolar dilution according to the following formula. We recommend the use of PBS without Ca²⁺ and Mg²⁺.

                 (D`- D%) *10<sup>2</sup>                      D`     required final density (g/ml)

V(%) =   ------------------------          D``    high starting density (g/ml)

                  D`` - D%                 D%   Density of the iso-osmotic dilution solution (g/ml)

                                               V(%)  Volume per cent of the starting solution with high density

The reactivity and stability of Ficoll^TM is based on its hydroxyl groups and on the glycoside bonds within the sucrose residues. It is stable in alkaline and neutral solutions. At pH values lower than 3, it is rapidly hydrolysed, especially at elevated temperatures. In neutral solutions, however, Ficoll^TM can be sterilised by autoclaving at 110°C for 30 minutes, without affecting the reactivity. Avoid heavily oxidizing or reducing substances.  

The Lymphocyte Separation Medium LSM 1077 is stable for 2 years when stored at +15°C to +25°C.

Note:
The Lymphocyte Separation Medium LSM 1077 is light sensitive. The medium should be protected from light during shipping and storage.

FICOLL is a trademark of GE Healthcare companies.