|
| |
Saponin/formaldehyde protocol (after
Jung et
al.)
Chemicals:
 |
Brefeldin A (BFA) |
|
|
Bovine Serum Albumin (BSA) |
|
|
Formalin (37%, methanol stabilized), dilute with PBS to 3.7% when needed. |
|
|
Ionomycin |
|
|
Lipopolysaccharide (LPS) |
|
|
Phorbol 12-myristate 13-acetate (PMA) |
|
|
Phosphate Buffered Saline (PBS) |
|
|
PBA; PBS / 1% BSA / 0.05% NaN3 |
|
|
Saponin |
|
|
Staphylococcus Aureus B (
SEB)
|
|
|
Antibodies (tested for intracellular staining)
|
1. Stimulate 2.106 PBMC/24 well plate well with:
|
|
10 ng/ml PMA, 1 µg/ml ionomycin
in the presence of 10 µg/ml BFA for 6 hours; to stimulate every cell type. |
|
|
1 ng/ml SEB for 6 hours, last 5 hours in the presence of 10 µg/ml
BFA; to stimulate T cells in the presence of APC. |
|
|
100 ng/ml LPS for 6 hours, last 5
hours in the presence of 10 µg/ml BFA; to stimulate monocytes or
monocyte derived dendritic cells. |
All
incubations/stimulations at 37°C, 5% CO2.
2. Harvest cells and wash once with PBS, 10 min.
300 g.
3. Fixate cells with 1.0% formalin at RT for 15 min.
4. Wash cells once with PBS, followed by one wash
with PBA, 10
min. 300 g.
5. Add 500 µl to the pellet and split sample over
(flow cytometer)
tubes (2.105 cells/tube/50 µl).
6. Prepare
saponin/antibody mixture: per sample you should add
50 µl mixture containing the antibodies and
the volume is
completed with 0.5% saponin/PBA.
7. Add 50 µl
saponin/antibody mixture and incubate at RT 30 min.
8. Wash sample with saponin buffer, 10 min 300 g.
9. Wash sample with PBA, 10 min 300g.
10. Add 200 - 400 µl PBA to
pellet.
11. Evaluate on flow
cytometer.
|
Protocol
