Two-step method for isolation of inactivated CD4+ T-cells from human blood mononuclear cells

Preparation of PBMC (peripheral blood mononuclear cells)

Collect at least 5mL of human whole blood to the

Store tube upright at Room temperature until centrifugation.

1500-1800rcf,18-25°C,0h 0m 0s in a horizontal rotor (swing-out head) for a minimum of 0h 15m 0s

Aspirate approximately half of the plasma without disturbing the cell layer.

Collect cell layer with a Pasteur Pipette and transfer to a 15mL size conical centrifuge tube

with cap.

Preparation of the Isolation buffer Isolation buffer

supplemented with 0.1% BSA and 2millimolar (mM) EDTA.

Preparation of magnetic particles

Resuspend the in the vial (i.e. vortex for > 0h 0m 30s or tilt and rotate for 0h 5m 0s)

Transfer the desired volume of to a tube.

Add the same volume of Isolation Buffer , or at least 1mL and resuspend.

Place the tube in a magnet for 0h 1m 0s and discard the supernatant.

Remove the tube from the magnet and resuspend the washed in the same volume of Isolation Buffer as the initial volume of Dynabeads®.

Isolation Procedure

Transfer 100µL (5 × 10⁷) PBMC in Isolation Buffer to a tube.

Resuspend the bead-bound cells thoroughly by pipetting >10 times using a pipette with a narrow tip opening. Avoid foaming.

Place the tube in the magnet for 0h 2m 0s. Transfer the supernatant containing the untouched human CD4+ T cells, to a new larger tube.

Add 2mL Isolation Buffer to the tube containing the Dynabeads^® and resuspend the bead-bound cells by pipetting as described in step 4.10.

Place the tube in the magnet for `0h 2m 0s` and then combine the two supernatants.

Add 20µL

Add 20µL of Antibody Mix

Mix well and incubate for 0h 20m 0s at 2-8°C

Wash the cells by adding 2mL Isolation Buffer . Mix well by tilting the

tube several times and 350x g,2-8°C. Discard the supernatant.

Resuspend the cells in 100µL Isolation Buffer .

Add 100µL pre-washed Dynabeads^®.

Incubate for 0h 15m 0s at Room temperature with gentle tilting and rotation

Add 1mL Isolation Buffer .

Preparing buffers for operations

• Buffer 1 : supplemented with 0.1% bovine serum albumin (BSA), 7.4 • Buffer 2 : with 0.1% BSA and 0.6% sodium citrate or 2millimolar (mM) EDTA.

Prepare Release Buffer

1. For each vial of freeze-dried DNase I, transfer 300µL from the Releasing Buffer Component

II to each tube of Releasing Buffer Component I (DNase I).

2. Aliquot the reconstituted Release Buffer into suitable portions. Store at -20°C. Thaw immediately before use and keep on ice once thawed. Thawed Release Buffer can be re-frozen once.

Wash Dynabeads‱ ^®

Resuspend the Dynabeads® in the vial (i.e. vortex for >0h 0m 30s or tilt and rotate for 0h 5m 0s. Transfer the desired volume of Dynabeads® to a tube (25µL for one sample).

Add the same volume of Buffer 1 , or at least 1mL and resuspend.

Place the tube in a magnet for 0h 1m 0s and discard the supernatant.

Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume of Dynabeads®.

Isolate Cells (Labeling Cells with Biotinylated Antibodies)

Add ~10µg primary biotinylated antibody to 1mL cell suspension and mix

Incubate for 0h 10m 0s at 2-8°C

Wash the cells by adding 2mL Buffer 2 and 350x g. Discard the supernatant.

Suspend the cells in 4mL Buffer 2 .

Add 25µL pre-washed and resuspended Dynabeads^®

Incubate for 0h 20m 0s at 2-8°C with gentle tilting and rotation.

Place the tube in the magnet for 0h 2m 0s. Transfer the supernatant containing the inactivated human CD4+ T-cells to a new larger tube.

Add 4mL Buffer 2 to the tube containing the Dynabeads^® and repeat step 8.7

Combine the two supernatants.