|
| |
Saponin/formaldehyde protocol (after Jung et
al.)
Chemicals:
 | Brefeldin A (BFA) |
| Bovine Serum Albumin (BSA) |
| Formalin (37%, methanol stabilized), dilute with PBS to 1.0% when needed. |
| Phosphate Buffered Saline (PBS) |
| PBA; PBS / 1% BSA / 0.05% NaN3 |
| Saponin
|
| Antibodies; tested for intracellular staining and try to prevent working
with biotinylated antibodies that require the use of (strept)avidine.
|
1. Wash the cells twice with PBS, 10 min.
300 g.
2. Fixate cells with 1.0% formalin at RT for 15 min.
3. Wash cells once with PBS, followed by one wash
with PBA, 10 min. 300 g.
4. Add 500 µl to the pellet and split sample over
(flow cytometer) tubes (2.105 cells/tube/50 µl).
5. 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.
6. Add 50 µl
saponin/antibody mixture and incubate at RT 30 min.
7. Wash sample with saponin buffer, 10 min 300 g.
8. Wash sample with PBA, 10 min 300g.
9. Add 200 - 400 µl PBA to
pellet.
10. Evaluate on flow
cytometer.
|
Protocol
