(i) 10 ml (EDTA) blood for DNA isolation for HLA class II analysis.

(ii) 10 ml (heparinised) blood for cell surface and intracellular cytokine staining

(iii) 5 ml for serum. The serum must be stored at –70° C.

1. Lymphocyte isolation : If protocol is not available, detailed protocol can be made available on request.
2. Intracellular staining and cell surface staining (see Annexure I).

For further details contact:

Preparation of reagents:

PBS: 1.9mM Na H₂PO₄, 8.1 mM Na₂HPO₄, 0.154 M NaCl. Set pH 7.2 -7.4 with 1M NaOH or 1M Hcl.

PMA: (2ng/ ml) Dissolve 10 mg in 500 m l DMSO and store the stock in aliquots at -20° C. Without repeated freezing or thawing, dilute 5 m l (100m g) of the stock with 95 m l PBS and store at -20° C. Dilute 1 m l of this stock with 999m l of RPMI plus 10 % FCS and use 2m l / ml culture to obtain 2 ng/ml final concentration.

Ionomycin: (2 m M) Prepare 5 mg/ml stock in DMSO (10mM). Dilute 200m l with 800 m l PBS (2mM) and use 1m l in 1 ml culture to obtain 2m M final concentration.

Monensin: (2 m M) Dissolve Monensin in absolute alcohol as a stock of 2mM and add NaOH to final 1 mM concentration. Use 1m l in 1 ml culture to obtain 2m M final concentration.

PBS+BSA+Azide buffer: Mix 500 ml PBS + 0.5 ml 1M Sodium azide in water + 2.5 gm BSA (Fraction V). Dissolve and filter sterilize. Store at 4° C

Freezing mixture: 10 % DMSO in 1% BSA (prepared in PBS).

4 % formaldehyde: Take 10.8 ml of 37.5 % formaldehyde from the bottle and make up the vol to 100 ml with PBS. Filter and store at 4° C.

1. Isolate lymphocytes from peripheral blood using ficoll gradient centrifugation.
2. Culture 2 x 10⁶ cells / ml of RPMI 1640 + 10%FCS or human AB serum preferably in 24 well plates. Add PMA (2 ng/ml) plus Ionomycin (2 m M) plus Monensin (2 m M) at 0 hrs and incubate at 37° C in 5% CO₂ incubator precisely for 6 hrs.
3. Harvest the cells in a 5 ml polystyrene tube and centrifuge at 1000 rpm for 10 minutes at room temperature for washing.
4. Wash twice with PBS+BSA+Azide buffer.
5. Wash once with PBS alone.
6. Resuspend the pellet in 200m l of 4% formaldehyde and add 200m l of PBS. Mix gently and place in dark at room temperature for 30 minutes.
7. Pellet the cells.
8. Wash once with PBS+BSA+Azide buffer.
9. Pellet the cells and resuspend in prechilled freezing mixture [1 % BSA + 10 % DMSO in PBS].
10. Immediately transfer to –70° C and store until shipped to the central laboratory for antibody staining and analysis. Shipment should be done in dry ice and ensure while transferring from –70° C to dry ice that cells don’t get thawed.