Materials:

• Equipment

  • 10-100 ml Pipette, Eppendorf

  • Rotixa/RP centrifuge, Hettich

  • Biosafety cabinet, Clean Air

  • FACStar, Becton Dickinson

• Disposables

  • Pipettes: 2, 5, 10 ml, Falcon, BD

  • Pipette tips: 1-200 ml, Costar

  • Propylene conial tube: 15 ml, Falcon, BD

  • Polypropylene round-bottom tube with cap: 6 ml (12x75mm), Falcon, BD

  • Bio-freeze vial: 6 ml (12x75mm), Costar.

• Reagents

  • Bovine serum albumine (BSA), Sigma

  • Phosphate buffered Saline (PBS)

  • Wash buffer (PB): PBS/1.0%BSA

  • RPMI-1640 with Hepes, Gibco BRL

  • Fetal bovine serum (FBS), Hyclone

• Antibodies

  • Leu2a-PE (g1;CD8), Becton Dickinson.

  • Leu3a-FITC (g1;CD4), Becton Dickinson.

Methods

• Thaw PBMC gently in 10 ml RPMI with 20% FCS, spin down (4°C, 5', 2000 rpm)

• Resuspend in 2 ml PB, take 40 ml in 20 ml Isoton for cell counting

• Add another 3 mL of PB and spin down (4°C, 5', 2000 rpm)

• In the mean time count the cells with the Coulter Counter

• Resuspend cells in PB at concentration to 10⁷ cells/mL

• Add directly labelled mAb to final concentration of 1:50

• Incubate 30' at 4°C in the dark.

• Wash twice with PB

• Resuspend cells in PB to 3*10⁶/mL in 6 mL tubes. (Max. 4.5 mL)

• Deliver also tubes with 0.5 mL RPMI/20% FCS for harvesting sorted cells

• Sort on FACStar approximately 1-2*10⁶ cells per T-cell subset

• Gate on lymphocyte population in dot plot

• Sort cells with high expression of CD4 or CD8 separately

• Analyze purity of T-cell subsets by analyzing 5,000 events

• Transfer sorted cell suspension to 15 mL conial tube

• Spin cells down (4°C, 5', 3000 rpm)