Phytohaemagglutinin (PHA) stimulation of cells

Reagents

PHA Stimulation Medium:

78ml RPMI 1640 (Dutch Modification) 20ml Heat inactivated FCS 1ml Penicillin/Streptomycin 1ml Glutamine

Washing Medium:

100ml RPMI 1640 1ml Penicillin/Streptomycin

0.1% Trypan Blue

Purified PHA (Murex Diagnostics):

HA15 10mg/ml stock. Add 4.5ml to 45mg vial and aliquot in 100m l amounts. Store at -20C.HA16 2mg/ml stock. Add 1ml SDW to 2mg vial and aliquot in 20m l amounts. Store at -20C.

Preparation of Cells

NOTE: All procedures should be carried out in a Biomat2 Cabinet or equivalent.

Monday and Friday are the most convenient days for preparing PHA blasts. The blasted cells must be harvested after 48-72 hours.

  1. Cells to be blasted with PHA should be resuspended at a concentration of 1x106 cells/ml in coculture medium. If the cells have been frozen then wash the cells twice by adding 10ml of Wash medium and centrifuging at 1200rpm for 10 minutes. Supernatants should be discarded into 5% Virkon. Perform a viability count using 0.1% trypan blue and adjust cell concentration accordingly.
  2. Cells are cultured in 10ml volumes at a concentration of 1 x 106 cells/ml using small tissue culture flasks.
  3. Either add 20ml of PHA (HA16 stock) to each 10ml culture (final concentration = 4-mg/ml).
    Or 10ml of PHA (HA15 stock) to each 10ml culture (final concentration = 10mg/ml)
  4. Incubate the culture flasks upright at 37C in a 5% CO2 incubator.