Negative Immunomagnetic Selection: A Method to Purify B-cells from Peripheral Blood Mononuclear Cells
Transcripción
Peripheral blood mononuclear cells or PBMCs, include B cells, T cells, natural killer or NK cells, and monocytes. B cells produce immunoglobulin molecules to fight infection. In leukemia, these cells multiply and metastasize to different organs.
To isolate B cells, begin with a suspension of isolated PBMCs. Add primary antibody mixture directed against specific cell surface molecules called cluster of differentiation or CD. Specific antibodies bind to the CD-3 molecule on T cells, CD-14 molecule on monocytes, and CD-16 molecule on NK cells, leaving the B cells unlabeled.
Next, wash the sample to remove the unbound antibodies. Add secondary antibody-conjugated magnetic microbeads to the sample. Secondary antibodies bind to the FC region of primary antibodies attached to specific cells. Place the tube containing the labeled cell suspension under the influence of a magnetic field. In the field, the magnetically labeled cells migrate and bind to the tube wall, leading to the negative selection of the unwanted cells. Collect the supernatant containing enriched B cells into a fresh collection tube.
In this protocol, we will perform a negative immunomagnetic selection of leukemic B cells from PBMCs of chronic lymphocytic leukemia patients.
Discard the supernatant and resuspend 10 million PBMCs in one milliliter of isolation buffer, which is PBS with 0.1% BSA and 2 millimolar EDTA, with the pH adjusted to 7.4. CLL lymphocytes now need to be purified from the PBMC preparations. Then, add 10 micrograms of mouse monoclonal anti-CD-3, CD-14, and CD-16 primary antibodies, and incubate for 30 minutes at 4 degrees Celsius.
After washing the cells with the isolation buffer and centrifuging at 500 times g for 5 minutes at 4 degrees Celsius, resuspend the 10 million PBMCs in 1 milliliter of isolation buffer. Incubate the resuspended cells with 100 microliters of washed magnetic beads for 30 minutes at 4 degrees celsius with gentle resuspending. Then, place the tube in the magnet holder for two minutes and transfer the supernatant with the unbound cells to a fresh tube with a 5 milliliter pipette. After this step, purified CLL lymphocytes are ready to be used.
