Con A activation of T cells

Goals and Rationale

Polyclonal stimulation of mouse T cells using Concanavalin A in vitro. This results in activation, proliferation and differentiation of both CD4 and CD8 T cells.

Con A is a lectin which non-specifically activates mouse T cells. It has little effect on B cells. T cell stimulation is rapid and the protocol is optimized for maximal proliferation at ~day 2 after stimulation. Longer term stimulation can be used to produce differentiated effector cells (e.g. CTL effector function appears at day 4-5), but input numbers might need to be reduced to prevent crashing of the culture.

Safety Concerns

Caution: This protocol uses Concanavalin A which is a biohazard - Follow the specific handling procedures and observe MSDS recommendations. Autoclave residual concentrated Con A stocks.

Methods

Ø Mouse spleen cells are prepared as usual into HBSS. Lymph node cells can also be used. Lyse red blood cells with ACK (this may not be necessary but it typically used) for 5 minutes. Add RP10 media and centrifuge. Resuspend in 5 mls of (warmed) RP10 media, count, and adjust the concentration to 2.5 x 10⁶ cells/ml.

Ø Thaw Con A stock vials (1 mg/ml) sufficient for the experiment.

Ø Aliquot 10 ml of cell suspension into a 25 cm² tissue culture flask. Add freshly thawed Con A to a concentration of 2.5 mg/ml or 5 mg/ml (i.e. 25ml or 50ml Con A stock per flask). It is best, if possible, to set up flasks at both concentrations of Con A to correct for variability in stimulation.

Ø Stand the flask ON ITS END (i.e. not laid flat but with the loosened cap pointing upwards) in the tissue culture incubator. This is to increase cell-cell contact during stimulation.

Ø Cells proliferate over the next 2 days. Keep an eye on the culture, it should gradually turn yellow as the cells acidify the media, but if it changes color rapidly, this may be contamination. Cell clumps should be very evident from 24 hours onwards. Be careful resuspending the cells before 48 hours as they may be fragile.

Ø At desired times, remove the activated cells and analyze. For functional assays (e.g. CTL activity), it may be necessary to inactivate residual Con A. This can be done by resuspending the cells in a-Methyl Mannoside (50mM in RP10; stock is 20x). a-MM blocks Con A. Cells can be used immediately after addition of a-MM.

Alternatives and Comments

Ø Cells can also be stimulated in plates (24-well and 96-well plates have been used in experiments). Similar cell densities and doses of Con A can be used.

Ø Cell densities may need to be reduced if TCR transgenic samples are used (if there are higher percentages of T cells in the sample), to avoid the culture crashing.

Reagents

Concanavalin A (also called Con A)

Calbiochem #234567: Mol.Wt. 104,000 (but exists as multimer)

Stock solution (100x – 200x) is 1mg/ml:

Ø Weigh aliquot of Con A powder into a 50ml centrifuge tube (AVOID BREATHING OR SPILLING POWDER – See MSDS sheets). Aim for around 10mg, but determine exact quantity.

Ø Add* sterile ddH₂O to give approximately 1.2 mg/ml (so, for 12 mg Con A, add 10ml water).

Ø Gently mix and let stand at 4^oC for at least 2 hours. Then adjust volume* with ddH₂O to give solution of 1mg/ml

Ø Con A does not go completely dissolve and so the solution will be hazy. Some Con A may therefore be lost in sterile filtration. In short term experiments, we have used Con A stocks which are NOT filtered, but if desired, sterilize through a 0.2mm filter.

Ø Store at –20^oC in 100ml - 1ml aliquots, depending on application.

a-Methyl Mannoside (also called Methyl-a-D-Mannopyranoside, or a-MM)

Calbiochem #462711: Mol.Wt. 194.2

Stock solution (20x) is 1M:

Ø Dissolve 194.2g a-MM into 800ml RPMI. Once in solution, adjust volume* to 1L.

Or, for small scale…

Ø Dissolve 9.71g a-MM into 40ml RPMI. Once in solution, adjust volume* to 50ml.

Ø Sterilize through 0.2 mm filter. Store 25 ml aliquots at -20^oC.

*Remember to use tissue culture grade plastic/glassware for preparing these solutions.