Separation of T-Cell Subsets by FACStar
10-100 ml Pipette, Eppendorf
Rotixa/RP centrifuge, Hettich
Biosafety cabinet, Clean Air
FACStar, Becton Dickinson
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.
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
Leu2a-PE (g1;CD8), Becton Dickinson.
Leu3a-FITC (g1;CD4), Becton Dickinson.
Thaw PBMC gently in 10 ml RPMI with 20% FCS, spin down (4Â°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 4Â°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 (4Â°C, 5', 3000 rpm)