Con A Stimulation

Procedure

1. Resuspend a (50%confluent to 100%confluent) 100mm Dicty plate.
2. Count Cells.
3. Make 5ml 30ug/ml ConA+ Mes Buffer (15ul 10mg/ml in 5ml of 20mM MES/2mM MgCl2/ph6.8)
4. Use cell count to determine volume needed for 3x10^6 cells.
5. Add HL-5 to two 60mm plates (enough to equal 3ml with addition of cells).
6. Add 100ul of TCA(100%) to two small 1.5ml microtubes. Label ConA+ and ConA-.
7. Add cells (voulme for 3x10^6 calculated above). Allow to adhere to plate for 5 min. Aspirate off media.
8. Add 3ml of MES buffer (the type described above!) and wash lightly. Aspirate off.
9. Add 2.5ml of MES Buffer. ConA+ to one plate and ConA- to the other. Incubate for 4min.
10. Resuspend w/ pipettor and move 1ml to TCA+ tubes. Freeze.
11. Add 2X urea sample buffer and run on a urea gel.