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)

Department of Microbiology
School of Medicine
University of Washington
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