IsolationofPBMCsfrompatientbloodandbuffycoats1

1| Transfer heparinized venous blood of patient to plastic50-ml tubes and dilute with an equal volume of PH buffer.

When sodium citrate has been used as anticoagulant, add an equal volume of PT buffer. When PBMCs are isolated from a buffy coat, transfer the buffy coat to a 250-ml flask and add PT buffer to a final volume of 150 ml.

2| Gently load 25 ml of diluted blood on top of 12.5 ml of a Ficoll-Isopaque solution with a density of 1.077 g ml
1 in a 50-ml tube.

3| Centrifuge at 760g for 20 min at room temperature (21 ± 5 1C). Slow acceleration and do not use brake.

4| Collect the cell band on top of the Ficoll layer with a Pasteur pipette and transfer it to a new 50-ml tube (maximum of 2 cell bands per tube) and add PH or PT buffer till a final volume of 50 ml. Centrifuge at 425g for 15 min at room temperature.

5| Wash the harvested cells twice in a total volume of 50 ml of PH or PT buffer and centrifuge at 425g for 10 min at room temperature.It is important to completely resuspend the cell pellet for each wash to ensure thrombocyte removal (see also below).

6| Resuspend the cells in IF medium and count the cells using an automated cell counter.

CAUTION Working with primary cells from blood donors and HIV-1 infected patients may result in the frequent introductionof mycoplasma. Precautions should be taken to prevent infection of other cells or cell lines. It is recommended to add ciprofloxacin to the cultures, to inhibit replication of mycoplasma.

CRITICAL STEP The presence of thrombocytes in the PBMCs cultures will have a negative effect on isolation of HIV-1. When the supernatant is not clear after the second wash (Step 5), large amounts of thrombocytes are still present in the PBMCs, and additional wash steps (repeat Step 5) should be added to remove the thrombocytes.

PAUSE POINT PBMCs can now be either cryopreserved in IMDM containing 10% (vol/vol) FCS and 10% (vol/vol) DMSO or directly used. Cryopreserved PBMCs can be stored in liquid nitrogen for more than 10 years.

Determination of a 32 bp deletion in the CCR5 gene of PBMCs donors

7| Isolate genomic DNA from 1  106 PBMCs or 200 ml of blood using the Qiagen blood kit or equivalent alternative,according to the manufacturer’s instructions.

8| Prepare the PCR mix as follows using primers CCR5-sense (position 427 to 450 in CCR5 (NM_001105536): 5￠-GATAGGTACCTGGCTGTCGTCCAT-3￠) and CCR5-antisense (position 665 to 644 in CCR5 (NM_001105536): 5￠-ACCAGCCCCAAGATGACTATCT-3￠)

flanking the described 32-nt deletion in the CCR5 gene.

Reagent Amount (ll)

PCR reaction buffer (10) 5

dNTP (5 mM) 2

MgCl (50 mM) 2.6

Primer CCR5-sense (100 ng ml
1) 1

Primer CCR5-antisense (100 ng ml
1) 1

Taq DNA polymerase 0.2

H2O 33.2

Total volume 45

9| Add 5 ml (10–100 ng) of genomic DNA to the reaction.

10| Amplify the PCR products under the following conditions in a thermocycler:

Step Temperature (1C) Time Number of cycles

1 95 5 min 1

2 95 5 s 35

55 10 s

72 1 min

3 72 2 min 1

4 4 N 1