Sample Purification

3.1.1. Sample Processing

1. Carefully overlay 20 mL blood onto 20 mL histopaque and spin in an un-braked centrifuge for 30 min at 400g.

2. Remove the lymphocytes which form a layer at the interphase between the histopaque and the plasma using a plastic Pasteur pipet and transfer to a clean tube.

3. Remove the granulocytes which form a layer on top of the red blood cell pellet underneath the histopaque at the base of the tube using a plastic Pasteur pipet and transfer to a clean tube.

4. Wash the lymphocytes with 30 mL PBS and the granulocytes with 30 mL water (to lyse the red blood cells).

5. Pellet cells, decant supernatant, and repeat this washing step.

6. Re-suspend the cells in residual fluid after decanting the second time.

7. Make a 1/100 dilution in PBS of the lymphocytes and count the number of cells either by using a haematology analyser such as an Advia 120 (Bayer, UK) or in Neubauer counting chamber. The lymphocytes are now ready for purification by either of the two suggested methods.

8. Transfer a small volume (20 |L) of the granulocyte suspension to a 1.5 mL micro-centrifuge tube in preparation for genomic DNA extraction. The remaining granulocyte pellet should be stored at -20┬░C.

Refer to the manufacturer's instructions on how to sort specific populations on the chosen machine. The sorted cells are analyzed by flow cytometry to verify the purity of the separation (Fig. 2).

3.1.3. Magnetic Micro-Bead Purification

Miltenyi CD19 micro-beads are supplied with a detailed protocol sheet, which is simple and straightforward. We access the purity of our samples by labelling 1 x 106 cells, both before and after the column purification, with CD5 FITC and CD20 RPE mouse antihuman monoclonal antibodies (Dako, Denmark) and analysing by flow cytometry (see Note 5).

We recommend the micro-bead purification method because it is less laborintensive and the yield is higher; however, the limitation is that it is difficult to sort on more than one parameter at a time.

Whole Blood Facs Plot

Fig. 2. FACS plots of peripheral blood from SLVL patient, (A) forward scatter versus side scatter of whole blood (B) whole blood labeled with k and X light chain mouse monoclonal antibodies (Dako, Denmark) before FACS sorting; (C) the same cells as in (B) but after FACS sorting for the clonal X population.

Fig. 2. FACS plots of peripheral blood from SLVL patient, (A) forward scatter versus side scatter of whole blood (B) whole blood labeled with k and X light chain mouse monoclonal antibodies (Dako, Denmark) before FACS sorting; (C) the same cells as in (B) but after FACS sorting for the clonal X population.

Was this article helpful?

0 0

Post a comment