Protocol

Cell source Stimulation Secretion stop Fixation Permeabilisation Staining Protocol

 

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Saponin/formaldehyde protocol (after Jung et al.)

Chemicals:

bullet Brefeldin A (BFA)
bullet Bovine Serum Albumin (BSA)
bullet Formalin (37%, methanol stabilized), dilute with PBS to 3.7% when needed.
bullet Ionomycin
bullet Lipopolysaccharide (LPS)
bullet Phorbol 12-myristate 13-acetate  (PMA)
bullet Phosphate Buffered Saline (PBS)
bullet PBA; PBS / 1% BSA / 0.05% NaN3
bullet Saponin
bullet Staphylococcus Aureus B ( SEB)
bullet Antibodies (tested for intracellular staining)

1.      Stimulate 2.106 PBMC/24 well plate well with:

bullet

10 ng/ml PMA, 1 g/ml ionomycin in the presence of 10 g/ml BFA for 6 hours; to stimulate every cell type.

bullet

1 ng/ml SEB for 6 hours, last 5 hours in the presence of 10 g/ml BFA; to stimulate T cells in the presence of APC.

bullet

100 ng/ml LPS for 6 hours, last 5 hours in the presence of 10 g/ml BFA; to stimulate monocytes or monocyte derived dendritic cells.

All incubations/stimulations at 37C, 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 l to the pellet and split sample over (flow cytometer)
       tubes (2.105 cells/tube/50 l).
6.    Prepare saponin/antibody mixture: per sample you should add
       50 l mixture containing the antibodies and the volume is
       completed with 0.5% saponin/PBA.
7.    Add 50 l 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 l PBA to pellet.
11.  Evaluate on flow cytometer.

 

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 Suggestions, literature and protocols are welcome at icstaining@schuitemaker.info. Copyright 2000 - 2005.
Page was last updated: 05-03-2005