Density Gradient Centrifugation: A Method to Isolate CLL Cells from Peripheral Blood

1. Isolation of CLL Cells from Patient Blood Samples

1. Peripheral blood samples from previously consented CLL patients are received from the clinic in EDTA blood collection tubes, accompanied by the white cell count (WCC). Purify the peripheral blood CLL samples according to the WCC. For WCC < 40 x 10⁶ cells/mL, proceed to step 1.1.1; for WCC ≥ 40 x 10⁶ cells/mL, proceed to step 1.1.2.
   1. Pour the contents of all the EDTA blood tubes into a 50 mL conical centrifuge tube and add 50 µL of Human B Cell Enrichment Cocktail per 1 mL of blood. Incubate at room temperature (RT) for 20 min. Proceed to step 1.1.2.
   2. Dilute the sample at a ratio of 1:1 with RT CLL wash buffer (phosphate-buffered saline (PBS), 0.5% Fetal Bovine Serum (FBS) and 2 mM EDTA).
2. Aliquot RT density gradient media into an appropriately sized conical centrifuge tube for the sample (10 mL into a 50 mL tube for 30 mL of sample or 4 mL into a 15 mL tube for 10 mL of sample).
3. Carefully layer the sample on top of the density gradient media and centrifuge at 400 x g for 30 min at RT.
   NOTE: Ensure that the centrifuge is at RT before the samples are placed in the centrifuge as a change in temperature will result in poor enrichment of mononuclear cells, and switch off the brake on the centrifuge, as sudden braking can disrupt the liquid interface.
4. Gently harvest the white layer of mononuclear cells that collect at the interface of the density gradient media and CLL wash buffer, into a fresh 50 mL conical centrifuge tube using a plastic Pasteur pipette.
5. Add 40 mL of CLL wash buffer to the isolated monolayer to wash the cells and centrifuge at 300 x g for 10 min at RT.
6. Discard the supernatant, resuspend the pellet by flicking the bottom of the tube, then repeat the wash step described in step 1.5.
7. Discard the supernatant, resuspend the pellet as described in step 1.6, then resuspend the pellet in a set volume of CLL wash buffer (up to 40 mL, depending on the size of the cell pellet).
8. Count the cells using trypan blue and a haemocytometer. Then proceed to flow cytometry to check the purity of CLL cells.
   NOTE: At this stage, the CLL cells can be cultured at a concentration of 10 x 10⁶ cells/mL in media to be used in experiments, and/or cryopreserved in 10% dimethyl sulfoxide (DMSO)/FBS for future work at concentrations of up to 100 x 10⁶ cells/vial.