B-cell depletion for T-cell enrichment

6.6 B-cell depletion for T-cell enrichment

6.6.1 Anti-immunoglobulin columns

MATERIALS AND EQUIPMENT Degalan V26 plastic beads

0.1 M phosphate buffer, pH 6.4 Phosphate-buffered saline (PBS)

10 mg purified rabbit anti-mouse immunoglobulin antibodies

METHOD

1 Wash 5 g of Degalan beads with distilled water, and then equilibrate with 30 ml phosphate buffer. Remove all liquid with a pipette.

2 Add 10 mg purified antibody (initial concentration 5 mg/ml).

3 Incubate at 45°C for 2 h, then at 4°C overnight.

4 Recover unbound antibody and calculate amount adsorbed to beads.

Control column Many cells passing down a column of protein coated onto plastic beads will stick non-specifically

because of the strong non-covalent intermolecular forces at the surface of the bead. Hence reten- tion of the cells will not only be related to the antiserum on the column. As a control for non- specific retention of cells a column of an irrelevant antibody, e.g. anti-keyhole limpet haemocyanin, must be prepared and used in an identical manner to that described for the anti- immunoglobulin column.

Cell fractionation

MATERIALS AND EQUIPMENT Mouse spleen or lymph-node cells Two 20-ml plastic syringe barrels Sintered plastic discs for columns Tissue culture medium containing 5% fetal bovine serum and ethylene diamine tetra-acetic acid

(EDTA), 5 m M Degalan beads coated with (a) anti-Ig and (b) an unrelated antibody

6.6B-CELL DEPLETION FOR T-CELL ENRICHMENT

METHOD

1 Pour the coated plastic beads into separate syringe barrels fitted with sintered plastic discs and equilibrate each with 30 ml of medium.

2 Seal off the column with a needle and rubber bung.

3 Incubate the column at 37°C for 30 min.

4 Cool to 4°C for 30 min before use.

5 Prepare a single-cell suspension of mouse spleen or lymph-node cells and deplete of

phagocytic cells. Wash in medium and adjust to 10 7 lymphocytes/ml.

6 Pipette 1 ml of lymphocytes onto each column.

7 Allow the cells to enter the column bed and reseal column.

8 Add 1 ml of medium to the column; allow it to enter the column. Under the conditions described the anti-Ig column will be able to deplete 10 aliquots of lymphocytes. The depleted population may be collected either as individual aliquots or as

a pool.

9 After the last aliquot of cells, wash the column through with 15 ml of medium. Collect effluent.

10 Concentrate the effluent cells by centrifugation and count in a haemocytometer.

TECHNICAL NOTES • The high non-specific retention by these columns can be minimized by a high flow rate. At a

flow rate of 2–3 ml/min about 20–30% non-specific loss may be expected. • All cell fractionation procedures must be carried out at 4°C. • T lymphocytes prepared by this technique may be contaminated by a variable proportion of

null cells (see Table 6.1).

Biomagnetic separation of cells Rapid separation of subsets of cells from a heterogeneous population has been very much sim-

plified by the development of Dynabeads. In the direct technique, magnetic beads are coupled to antibodies which target a specific cell-surface antigen. Once added to the heterogeneous mixture of cells, the beads bind to the target ligand. Cell-bound beads can be separated from rest of the suspension using a Dynal magnet particle concentrator. The indirect technique may be used to isolate the cell subset of interest indirectly by the removing other cell types from the heteroge- neous suspension of cells, i.e. depleting the cell suspension of unwanted subsets of cells.

6.6.2 General protocol for the isolation of cells using Dynabeads

MATERIALS Dynabeads M-450 coated with primary antibody (see Table 6.2) Dynal magnetic particle concentrator (Dynal MPC) Phosphate-buffered saline (PBS), pH 7.4

C H A P T E R 6: Isolation of cells

Table 6.2 Dynabeads available for the isolation of human immune cell subsets from whole blood, buffy coat or peripheral blood mononuclear cells

Dynabeads M-450 coated with

Leucocytes

anti-CD45

B lymphocytes

anti-CD19

T lymphocytes

anti-CD3, anti-CD2 anti-CD4, anti-CD8

Granulocytes

anti-CD16, anti-CD15

Haemopoietic progenitor cells

anti-CD34

Antigen-presenting cells

anti-HLA class II

Monocytes/ macrophages

anti-CD14

Natural killer cells

anti-CD56, anti-CD16

Dynabeads are also available for the isolation of murine immune cell types.

METHOD

1 Use 1 × 10 7 Dynabeads M-450 per ml of cell suspension. Gently vortex the Dynabeads, then allow to separate using the Dynal MPC.

2 Remove the liquid and wash three times with an equal volume of PBS.

Direct isolation of cells

3 Add the washed Dynabeads M-450 to the heterogeneous cell suspension which contains the cells to be isolated.

4 Incubate with gentle mixing at 4°C for 30 min.

5 Insert the tube containing the cells and Dynabeads M-450 into the Dynal MPC, and allow to stand for 5 min. Cells possessing the target cell’s surface ligand are rosetted on the side of the test tube.

6 Discard the supernatant and wash the rosetted cells in PBS (or cell culture media).

7 Insert the tube containing the cells and Dynabeads M-450 into the Dynal MPC, and allow to stand for 3 min.

8 Repeat steps 6 and 7 three times.

Indirect isolation of cells

9 Add the washed Dynabeads M-450 to the heterogeneous cell suspension which contains the cells to be isolated.

10 Incubate with gentle mixing at 4°C for 30 min.

11 Insert the tube containing the cells and Dynabeads M-450 into the Dynal MPC, and allow to stand for 5 min. Cells possessing the target cell’s surface ligand are within the supernatant.

12 Transfer the supernatant to another test tube.

6.6B-CELL DEPLETION FOR T-CELL ENRICHMENT

6.6.3 Nylon wool columns

Spleen-cell suspensions may be fractionated on the basis of their differential adherence to nylon fibres. At 37°C and in the presence of serum, B lymphocytes will bind avidly to nylon wool columns, giving an effluent population of virtually pure T lymphocytes and ‘null’ cells. This tech- nique has the obvious advantages of speed, convenience and low cost.

MATERIALS AND EQUIPMENT Nylon wool, sterile Tissue culture medium containing 5% fetal bovine serum (FBS) Syringe, 20 ml, plastic, sterile

METHOD

1 Pack 600 mg of sterile nylon wool (approximately 6 ml) into a 20-ml syringe barrel and wash with tissue culture medium containing 5% FBS.

2 Seal the column and incubate at 37°C for 1 h.

3 Prepare cells, depleted of phagocytic cells, and adjust to 5 × 10 7 lymphocytes/ml.

4 Flush column with 5 ml of warm tissue culture medium. (This will correct any change in pH during incubation.)

5 Add 2 ml of cell suspension dropwise to the top of the column. After it has all entered, add

1 ml of warm tissue culture medium.

6 Seal the column and incubate at 37°C for 45 min.

7 Wash the column with 25 ml of warm tissue culture medium and collect the unbound cells in the effluent.

8 Concentrate the cells by centrifugation (150 g for 10 min at 4°C) and determine the number of viable lymphocytes.

The effluent population should be depleted of B lymphocytes, as evidenced by anti-immunoglobulin immunofluorescence, and will consist of T lymphocytes and ‘null’ cells. A proportion of the bound cells may be recovered by mechanical elution as follows.

1 Wash the column with 100 ml of warm tissue culture medium and discard the effluent.

2 Seal the column with a needle and rubber bung.

3 Add 2 ml of warm tissue culture medium and squeeze the nylon wool with blunt stainless steel forceps.

4 Unseal the column and wash with 10 ml of warm tissue culture medium. Finally, replace the syringe piston and expel all the tissue culture medium.

5 Collect the effluent cells and concentrate by centrifugation (150 g for 10 min at 4°C).

6 Count number of viable lymphocytes/ml. The cells recovered by mechanical elution will consist of B lymphocytes, as evidenced by anti-

immunoglobulin immunofluorescence, contaminated with a variable number of T lymphocytes and ‘null’ cells.

C H A P T E R 6: Isolation of cells