Activation Assay Protocol for cytokine release

Activation Assay Protocol for cytokine release

Buffers/Reagents:
PBS-1%BSA
PBST (ELISA wash buffer)
   100mL 10X PBS
   900mL dH2O
   375uL Tween-20
Coat Buffer
   26.807g Na2HPO4-7H2O
   pH to 9.0

1.   If not coating plate for cross-linking, skip to step 6.
2.   For testing ability of clones to secret IL-2 through TCR stimulation use either anti-TCR Vbeta antibody or anti-CD3.
3.   Make up a final concentration of stimulating TCR antibody at 0.5ug/mL for 50uL per well in coating buffer, anywhere from 0.1 to 1.5ug/mL will give IL-2 release, but 0.5ug/mL is right around the concentration required for ? maximal IL-2 release and thus most useful in experiments looking for change in IL-2. However the user should titrate this to determine optimal concentration.
4.   Coat 50uL per well, incubate at least 2 hours or overnight.
5.   Block using PBS-1%BSA at least 30 minutes no more than 2 hours, 100uL per well.
6.   Count cells. Normally use 5x104 cells per well. Cells should be re-suspended well before counting to assure proper count.
7.   Trypan blue is not necessary to count cells, unless the user has a hard time distinguishing live from dead, which look completely different/
8.   Place 10uL of cell suspension or 10uL of cell/trypan blue suspension on hemacytometer.
9.   Count number of cells in each 16 square grid. Or count 2 squares and divide by 2. If using trypan blue divide by 2 and 4 respectively.
10.   Multiply this number by 104, this is how many cells per mL you have and is based on the volume in a small grid.
11.   Calculate usage by multiplying number of wells needed by number of cells needed per well.
12.   Take out appropriate volume of cells and spin down cells and decant supernatant.
13.   If coating plate, dump out coat and block solution and smack dry.
14.   Final volume in each well should be 200uL and should consist of 50uL effector cells, 50uL target cells if used, 50uL suspension of blocking antibody if used and 50uL suspension of peptide if used. If any are omitted, the remaining volume can be substituted with media.
15.   Blocking antibody concentrations should be determined for each antibody but are typically around 10ug/mL, peptide typically around 10-6
16.   Blocking antibody can be preincubated with cells prior to addition to wells, as can activating peptide.
17.   Peptide is usually pre-incubated with cells for about an hour at 37 degrees.
18.   Incubate plate at 37 C for 27-30 hours with T cell lines or for 4 hours to overnight for primary NK cells.
19.   Remove supernatant and place directly on coated ELISA plates. It is not necessary to centrifuge since cells will have settled to bottom.
20.   Plate can be frozen and stored at -20 C until ready to assay.