Equipment and reagents
• 96-well microtitre plates (Nunc, distributed by Gibco BRL)
• Glass-fibre filters to match the beta-counter (Pharmacia)
• Complete culture medium: RPMI-1640, 10% HS, 1% sodium pyruvate, 1% non-essential amino acids (NAA), 50 mM 2-mercapto-ethanol, 20 n-g/ml gentamicin
1. Take flat-bottomed, 96-well microtitre plates and coat with 50-100 m-I of anti-CD3 (5 ti,g/ml) and anti-CD28 (5 n.g/ml) in PBS for 2-4 h at 37°C.
2. Wash the plates thoroughly with complete culture medium by dispensing 100 p,l into each well and then removing it carefully by suction. Repeat this wash step once more and then dispense 100 |xl of the complete culture medium into the appropriate wells.
3. Resuspend the CD4 T-cell population under investigation at 1 x 106/ml and plate out 100 (¿l/well.
• Cell harvester (Skatron)
• Stimuli (mitogens or antibodies of choice)
• 3H-labelled methyl thymidine (Amersham) ([3H)thymidine stock at 1 mCi/ml; dilute to give a working stock of 50 (iCi/ml in complete medium)
4. Prepare triplicate wells for each stimulus. Set up control wells to contain cells without stimulus.
5. Incubate the cells for 1-4 days at 37 °C, 5% C02 (see variations below).
6. Add 10 nJ [3H]thymidine (0.5 (xCi/well) for the last 6 h of culture.
7. Measure the incorporated radiolabel by harvesting the cells on to glass-fibre filters using a cell harvester. Allow the filter to dry overnight at room temperature.
8. Once dried, seal the filter in a bag with 10 ml scintillant fluid (Wallac) and read using a beta-counter—the readout will give the counts per min (c.p.m.) of radioactivity in the samples.
4.1.1 Variations and problems
• Do not leave cells in the presence of the radiolabel for more than 6 h, as longer incubation times result in extensive thymidine breakdown.
• For CD4 T-cell effectors and clones, proliferation can be measured following a 24-h stimulation. Freshly purified CD4 T cells from PBMCs require a 3-4 days' culture.
• For anti-CD3 and PMA stimulation, coat plates with anti-CD3 as above and use PMA at a final concentration of 1 ng/ml.
• For PMA and ionomycin stimulation, make up a PMA solution as described in Protocol 6, step 2 and add ionomycin at a final concentration of 400 ng/ml.
• It is important to optimize the stimuli for optimal proliferation, as high doses of mitogen, e.g. anti-CD3, can induce a state of 'unresponsiveness' or cell death, thus compromising the proliferative response.
• For freshly isolated CD4 T cells plate cells at 105/well. For CD4 clones and effectors plate at 1-2 X 104 cells/well.
Protocol 9. Antigen-specific proliferation
Equipment and reagents
1. Use freshly isolated CD4 T cells from PBMCs or CD4-effector cells or clones on day 14 of the stimulation cycle (see Protocols 5 and 6). Wash the cells in complete culture medium (no IL-2) before use.
2. Prepare autologous PBMCs (feeder cells) at 1 x 106/ml in complete medium and irradiate at 4000 rads.
3. Plate out 100 jjil/well of CD4 clones/effectors (2 x 104/well), and 100 jjlI irradiated autologous PBMCs (feeders). Culture without antigen (control) or with antigen at the appropriate concentration in 96-well plates at 37°C, 5% C02. Prepare at least three wells for each dilution of antigen, and for the controls.
4. Incubate the cells for between 48 h and 4-6 days (see below). Add 10 jjlI [3H]thymidine (0.5 jjiCi) for the last 6 h of culture in complete culture medium.
5. Harvest the cells on to glass filters and count in a Matrix beta-counter (see Protocol 8).
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