TITLE: Two-step method for isolation of inactivated CD4+ T-cells from human blood mononuclear cells
AUTHORS: Anna Esman, Michael Vinokurov

[DESCRIPTION]
1. Obtaining human CD4+ T cells
2. Obtaining CD4+ inactivated cells

using 

1.   

2.   
3.   
4.   
5.   
6.   

and magnetic tube separator. 

[STEPS]
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5. Preparing buffers for operations
• Buffer 1:     supplemented with 0.1% bovine serum albumin (BSA), pH 7.4 
• Buffer 2:     with 0.1% BSA and 0.6% sodium citrate or 2 millimolar (mM) EDTA.

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1. Preparation of PBMC (peripheral blood mononuclear cells)
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2. Preparation of the Isolation buffer 
    supplemented with 0.1% BSA and 2 millimolar (mM) EDTA.


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3. Preparation of magnetic particles
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1.1. Collect at least 5 mL of human whole blood to the    

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1.2. Store tube upright at Room temperature until centrifugation. 
 

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1.3.  1500-1800 rcf, 18-25 °C in a horizontal rotor (swing-out head) for a minimum of 15 min 
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1.4. Aspirate approximately half of the plasma without disturbing the cell layer.
 

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

 

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4. Isolation Procedure
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3.1. Resuspend the  in the vial (i.e. vortex for > 30 s or tilt and rotate for 5 min)

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3.2. Transfer the desired volume of   to a tube.
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3.3. Add the same volume of Isolation Buffer, or at least 1 mL and resuspend.
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3.4. Place the tube in a magnet for 1 min and discard the supernatant.

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3.5. Remove the tube from the magnet and resuspend the washed  in the same volume of Isolation Buffer as the initial volume of Dynabeads®.
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4.1. Transfer 100 µL (5 × 107) PBMC in Isolation Buffer to a tube.

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4.2. Add 20 µL    

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4.3. Add 20 µL of Antibody Mix
 


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4.4. Mix well and incubate for 20 min at 2-8 °C 

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4.5. Wash the cells by adding 2 mL Isolation Buffer. Mix well by tilting the
tube several times and 350 x g, 8 min, 2-8 °C. Discard the supernatant.
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4.6. Resuspend the cells in 100 µL  Isolation Buffer.

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4.7. Add 100 µL pre-washed Dynabeads‱.
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4.8. Incubate for 15 min at Room temperature with gentle tilting and rotation

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4.9. Add 1 mL Isolation Buffer.
 

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4.10. Resuspend the bead-bound cells thoroughly by pipetting >10 times using a pipette with a narrow tip opening. Avoid foaming.
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4.11. Place the tube in the magnet for 2 min. Transfer the supernatant containing the untouched human CD4+ T cells, to a new larger tube. 

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4.12. Add 2 mL Isolation Buffer to the tube containing the Dynabeads‱ and resuspend the bead-bound cells by pipetting as described in step 4.10.
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4.13.     Place the tube in the magnet for 2 min and then combine the two supernatants.
 

 

        
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6. Prepare Release Buffer 
1. For each vial of freeze-dried DNase I, transfer 300 µL 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. 
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8. Isolate Cells (Labeling Cells with Biotinylated Antibodies)
    
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7. Wash Dynabeads‱
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7.1. Resuspend the Dynabeads® in the vial (i.e. vortex for >30 s or tilt and rotate for 5 min. Transfer the desired volume of Dynabeads® to a tube (25 µL for one sample).

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

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7.3. Place the tube in a magnet for 1 min and discard the supernatant.

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7.4. Remove the tube from the magnet and resuspend the washed Dynabeads® in the same volume of Buffer 1 as the initial volume of Dynabeads®.
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8.1. Add ~10 µg primary biotinylated antibody to 1 mL cell suspension and mix             

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8.2. Incubate for 10 min at 2-8 °C
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8.3. Wash the cells by adding 2 mL Buffer 2 and 350 x g, 8 min. Discard the supernatant.
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8.4. Suspend the cells in 4 mL Buffer 2.
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8.5. Add 25 µL pre-washed and resuspended Dynabeads‱
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8.6. Incubate for 20 min at 2-8 °C with gentle tilting and rotation.
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8.7. Place the tube in the magnet for 2 min. Transfer the supernatant containing the inactivated  human  CD4+ T-cells to a new larger tube.
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8.8. Add  4 mL Buffer 2 to the tube containing the Dynabeads‱ and repeat step 8.7
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8.9. Combine the two supernatants.