Protocol: Thymocyte Il-2/PHA Co-Stimulation Assay

This assay must only be performed by an authorised radioisotope user and safety procedures for 3H-thymidine must be followed in a designated radioactive area. This assay measures IL-2 and Phytohemagglutinin (PHA) induced proliferation of primary chicken thymocytes by incorporation of 3H-thymidine. To ensure proliferation is due to IL-2 and not other cytokines e.g IL-1ß, pre-incubation of samples with a neutralizing antibody is essential.

Reagents:

• Recombinant chicken IL-2
• Dulbecco’s Modified Eagle’s Medium
• Bovine Serum Albumin (BSA) Fraction V
• Phytohemagglutinin-P (PHA-P)
• Ficoll-Paque or Histopaque 1.077
• Mouse anti-chicken IL-2 monoclonal antibody (neutralizing) clone 4F12
• DMEM/BSA assay media (DMEM supplemented with 2mg/ml BSA, 1% L- glutamine, 1 U/ml penicillin and 1ug/ml streptomycin)
• 3H-thymidine

Assay Procedure:

1. Transfer collected thymus from sterile DMEM into sterile petri dish containing 15ml fresh DMEM. Remove connective tissue/fat and tease apart using forceps. Pipette up and down gently to obtain single cell thymocyte suspension and transfer to a universal. Layer cells over 5ml Ficoll-/Histopaque 1.077 in a 50 ml Falcon tube then centrifuge at 1350g for 20mins.
2. Wash cells collected from interface three times in DMEM by spinning at 750g for 10 mins. Discard the supernatant and resuspend the cell pellet for each spin. Perform viable cell count and resuspend at 2 x 107 cells/ml in DMEM/BSA
3. Set up control samples in triplicate wells of a U-bottomed microtitre plate:
   Positive control - perform 2-fold serial dilutions of rchIL-2 (PAP005) in DMEM/BSA to a final volume of 100 ul/well.
   IL-2 Negative control – Test samples require a 1 hour room temperature pre-incubation with an IL-2 neutralising monoclonal antibody (MCA1943Z), prior to the addition of the thymocytes in step 6. Recommended final concentration for MCA1943Z is 2-20 µg/ml in the test sample.
   Negative control - repeat above procedure on COS free growth media alone.
4. Repeat above procedure on test samples.
5. To demonstrate background proliferation of cells without PHA-P addition, plate out thymocytes at 100 µl/well (2 x 106 cells/well), in row containing growth media alone.
6. To remaining thymocytes add 4 µg/ml PHA-P (to give a final concentration of 2 µg/ml when 100 µl cells added to test wells). Plate out these thymocytes at 100 µl/well (2 x 106 cells/well) to each of the triplicate wells prepared for controls and samples.
   Please note: PHA-P may require initial titration to determine concentration for sub-optimal stimulation.
7. Incubate plates at 41°C, 5% CO2, for 72hours. Prepare 3H-thymidine ready for pulsing cells for the final 6hrs of incubation:
   Transfer enough 3H-thymidine for 0.037MBq/test well into a Bijoux or screw-top tube and add appropriate volume of PBS for 1:10 dilution (e.g 100 ul 3H-thymidine plus 900 ul PBS). Add 10 ul of this to each well after 66hrs of plate incubation. Place plates in labelled boxes with lids and incubate in designated incubator at 41 °C, 5% CO2, for final 6hours.
8. Harvest onto filter mats using automated cell harvester and air dry. Place air-dried filter mats into bags, add scintillant, seal bags and obtain count in beta-counter.

Results:

Can be expressed as:

1. Stimulation indices (SI), representing the ratio of mean CPM of the co-stimulated thymocytes to mean CPM of the PHA-P only stimulated thymocytes.
2. Plot of mean CPMs of samples against mean CPMs of controls.
   
   Since there is no international standard for recombinant chicken IL-2, the results cannot be expressed as U/ml (unless simply relating results to PAP005 used) nor as activity ng/ml, since PAP005 is not a purified protein.

References

1. Rothwell, L. et al. (2001). Production and characterisation of monoclonal antibodies specific for chicken interleukin-2. Vet. Immunol. Immunopathol. 83: 149-160.
2. Lawson, S. et al. (2000). Turkey and chicken Interleukin-2 cross-react in in vitro proliferation assays despite limited amino acid sequence identity. J. Int. Cyt. Res. 20: 161-170.