Protocol

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Saponin/formaldehyde protocol (after Jung et al.)

Chemicals:

bulletBrefeldin A (BFA)
bulletBovine Serum Albumin (BSA)
bulletFormalin (37%, methanol stabilized), dilute with PBS to 1.0% when needed.
bulletPhosphate Buffered Saline (PBS)
bulletPBA; PBS / 1% BSA / 0.05% NaN3
bulletSaponin
bulletAntibodies; 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 Ál to the pellet and split sample over (flow cytometer) tubes (2.105 cells/tube/50 Ál).
5.    Prepare saponin/antibody mixture: per sample you should add 50 Ál mixture containing the antibodies and the volume is completed with 0.5% saponin/PBA.
6.    Add 50 Ál 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 Ál PBA to pellet.
10.  Evaluate on flow cytometer.

 

 

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Page was last updated: 05-03-2005