I am looking for a protocol for the stimulation of Jurkat T cells with soluble anti-CD3 and ati-CD28 antibodies.
I am working on the T-cells stimulation and effects of on kinectics. In my protocol that I am currently using I take 6 million cells in 1ml complete media per tube, rest the cells for 30 mins. in the incubator and add the drug and incubate it for an hour. After the incubation I stimulate the cells with 10ug/ml CD3 antibodies at different time points (0.2, 1, 2 and 20 min.) and quickly centifuge the tube to stop the stimulation.
This is a protocol I've used for primary CD4+ T cells which you can adopt for Jurkats.
1. Coat 6-well plate w/ α-CD3: 10 ug/ml α-CD3 in PBS, 1 ml/well; incubate @ 37º C at least 30’, usually 60’-90’
2. Resuspend cells in STCM* 1 M/ml and add 1 ug/ml α-CD28 Ab.
3. When α-CD3 coating is done, wash 6-well plate 1 x 5 ml PBS, then add cells to each well – do not plate more than 5 ml/well (minimum is 2 ml).
4. For CD4+ T cells, incubation is usually done at 37º C for 3-4 days. Your incubation period may vary.
*STCM or super T cell medium is made by adding the following to 500 ml of RPMI: 55 ml of HI-FBS, 224 μl of IL-2 stock (for a final concentration 100 U/ml) and 5–10 ml of TCGF. (Kim et al., 2014)
In solution, we normally use 2ug/ml OKT3 and 1ug/ml anti-CD28 (clone 9.3) and 5ug/ml goat anti-mouse IgG to crosslink and get a maximal activation.
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