Density Gradient Centrifugation: A Method to Isolate CLL Cells from Peripheral Blood
Transcript
– In chronic lymphocytic leukemia, or CLL patients, B cell lymphocytes significantly increase in the bone marrow and blood. To isolate these CLL cells from a blood sample, first, treat the sample with a human B cell enrichment cocktail that separates B cells using negative selection. The antibodies in the cocktail specifically recognize and cross-link the antigens expressed by non B cells and erythrocytes, forming dense complexes.
Next, layer the sample over a density gradient medium having a density optimal for lymphocyte isolation. With subsequent centrifugation, the different blood components separate based on variations in their densities relative to the gradient medium. The RBC-non-B-cell complexes with higher densities settle down through the medium, forming a pellet. The least dense plasma fraction forms the topmost layer. The less dense CLL cells form a white layer at the plasma-density medium interface.
Carefully transfer the white layer into a fresh tube. Centrifuge again to obtain a pellet containing purified CLL cells. In the following protocol, we show the isolation of chronic lymphocytic leukemia cells, from patient blood samples.
– Obtain peripheral blood samples from previously consented chronic lymphocytic leukemia patients in EDTA blood collection tubes accompanied by the white cell count.
– Human blood samples should be regarded as hazardous, as they potentially contain bloodborne viruses. Please ensure that these samples are handled in a Class II Biosafety Cabinet and that the user is wearing a lab-coat and gloves at all times. Dispose of blood contaminated materials and disinfect these.
– If the white cell count is equal or greater than 40 million cells per milliliter, dilute the sample at a ratio of 1 to 1 with RT CLL wash buffer. Then aliquot RT density gradient medium into an appropriately sized conical centrifuge tube for the sample. Carefully layer the sample on top of the medium and centrifuge at 400 times G for 30 minutes at RT.
Using a plastic pasteur pipette, gently harvest the white layer of mononuclear cells that collected at the interface of the density gradient medium and CLL wash buffer. Transfer this isolated monolayer into a fresh 50 milliliter conical centrifuge tube. To wash the cells, add 40 milliliters of CLL wash buffer to this monolayer and centrifuge at 300 times G for 10 minutes at room temperature.
Discard the supernatant, flick the bottom of the tube to re-suspend the pellet, and repeat this wash. Discard the supernatant, flick the bottom of the tube again, and then re-suspend the cell pellet in a set volume of CLL wash buffer, depending on the size of the pellet. After counting the cells and after flow cytometry to check the cell purity, place the cells into tissue culture plates at the required cell density, ready for stimulation, or drug treatment.
