Isolation of B-cell subpopulations by immunomagnetic bead assay

Summary

Anti-CD 19 labeled B cells, which have relatively low signal intensity, show mitogenic and antibody secretory responses similar to those obtained by negative screening. To prevent possible activation of cells by positive selection, the F (al/)2 fragment of the antibody can be used. Author: J.E. Colligan et al, Translated by Xuitao Cao et al. This experiment is from the "Compendium of Immunology Laboratory Guidelines".

Operation method

Separation of B-cell subpopulations by immunomagnetic beads

Move

basic program Material

Anti-coagulated blood (Appendix 3G), or fresh PBM C or cryopreserved PBMC isolated from whole peripheral blood (Unit 8.1) or tissue (Unit 7.8) (Appendix 3B). Fresh PBM C or cryopreserved PBMC isolated from whole peripheral blood (Unit 8.1) or tissue (Unit 7.8) (Appendix 3B).

1 % FBS (heat inactivated) in cold PBS (Appendix 1, calcium free)

Immunomagnetic bead-coupled anti-CD 19 antibody (4 X ¹⁰⁸ cells/ml; Dynal)

Cold RPMI-IO complete medium

25 cm2 and 75 cm2 Cell Culture Bottles (as Coming)

15 ml and 50 ml polypropylene-chan tubes (e.g. Falcon)

Magnetic separators (Dynal MPCI or Perspective Diagnotics BioMag Separator) or strong magnets

Shaker, 4°C

Detach-a-bead (Dynal), optional

Ia. Treatment of anticoagulated blood: Dilute 5-50 ml of blood with 3 times the volume of PBS containing 1 % FBS in a 75 cm2 cell culture flask in an ice bath.

Ib. Treatment of Isolated PBMC: Resuspend cells in 15 ml polypropylene tubes with cold PBS containing 1 % FBS at a concentration of ⁷ to 2 X ¹⁰⁷ cells/ml (Appendix 3A) in an ice bath.

2 . Vigorously shake the bead-coated anti-CD1 9 into a 15 ml polypropylene tube. Add 4 to 10 beads per target cell (e.g., 5 to 12.5 ul of Dynerl beads per I ml of undiluted blood or PBMC to estimate the number of cells in whole blood).
cells/ml).

3 . Add IO ml of cold 1 % FBS, shake, and attract the beads to the wall of the tube with a magnetic device or strong magnet. Wait about 5 m in to allow the beads to gather close to the magnet and place the syringe tip at the bottom of the tube to aspirate the liquid.

4 . Repeat step 3, then resuspend the beads with 1 to 2 ml of cold 1% FBS and add the beads to a culture flask containing the blood sample (step la) or to a 15 ml centrifuge tube containing PBMC (step lb). Incubate on a shaker at 6 to lOr/m iri for 30 min at 4°C (this speed allows sufficient mixing of cells and beads without destroying the cells). A portion of the cells can be taken and viewed under a microscope to determine that the magnetic beads surround some cells and are not present around others.

5 . Separate the cells with the magnetic device (step 3). Collect unbound cells by placing the tip of a syringe at the bottom of the bottle or tube. Remove the bottle or tube from the magnetic field and resuspend the magnetic beads in IOml of cold 1% FBS.

6 . Repeat step 5 no less than 5 times. After the last separation, do not resuspend the beads.
Captivation to dissociate B cells bound to magnetic beads

7a. Resuspend bead-bound cells with IOml of cold RPMI-IO complete medium, transfer to 25 cm2 cell culture flasks and incubate overnight at 37°C, 5 % C. 02 incubator.

8a. Resuspend the cells in a 15 m l tube and magnetically detach the immunomagnetic beads (now detached from the cells) at room temperature. Aspirate the unbound cell suspension into a new 15m l tube. Repeat the separation to remove excess magnetic beads if needed.

9a. Centrifuge the cells at 150 g for lOmin at 4°C, discard the supernatant, and resuspend the cells at the appropriate concentration in ice-cold RPMI-IO Complete Medium (or other appropriate medium). Determine purity by flow cytometry (Unit 4.2).

Dissociate B cells with Detach-a-b e a d polyclonal antibody.

7b. Resuspend cells bound to magnetic beads in IOOuI cold R P M I -I O complete medium, transfer to a 15 ml tube, add 10 ul (I U) of Detach-a-bead, and incubate on a shaker (e.g., orbital shaker) at room temperature for 45-60 min. ensure that the cells settle to the bottom of the tube.

Detach-a-b ead is a polyclonal antibody that binds to the F a b fragment of the mouse monoclonal antibody, thus breaking the connection between the cells and the beads.

8b. Cells are resuspended in 7 ml of cold RPM I-10 complete medium and tubes are placed in the magnetic field to separate the immunomagnetic beads. Place the syringe tip at the bottom of the bottle or tube to collect the unbound cell suspension into a new 50 ml centrifuge tube in an ice bath. Repeat 2 or 3 times and pour the cell suspension into the same centrifuge tube.

9b. Centrifuge the cells at 150° C for lOmin at 4°C, discard the supernatant, resuspend the cells in RPMI-10 complete medium on ice (or other appropriate medium) and centrifuge again. Repeat washing one or two or more times, resuspend the cells in ice RPMI-10 complete medium at the appropriate concentration, and check for purity by flow cytometry (Section 4.2).

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