Intracellular staining of markers with fluorochrome-conjugated antibodies

Intracellular staining is more demanding than staining of surface-proteins. Additionally to the usual staining steps (staining, washing), the cells must be fixated and permeabilized.

1) Harvest of cells and washing:

Count the amount of cells using a counting chamber and adjust the cell density to 1 - 5 x 10⁶ /ml with ice-cold PBS (alternatively PBS with 10% FCS and 1% sodiumazide).
Usually, the cells are stained using 12 x 75 mm round-bottom polystyrene (FACS) tubes. These can be used immediately in your measurements.

2) Add cells:

Add the desired amount of cells to the tubes (e.g. 150 µL for 150.000 cells per condition with 1 x 10⁶ cells/ml).

3) Fixation:

Add 100 µL of fixation liquid (e.g. 4% parafomaldehyde) and incubate 15 min at 4°C. Wash the suspension in PBS, with 0.2% Tween 20 (appr. 5 min at 400g).

4) Permeabilization:

Add 100 µl of permeabilization-liquid (z.B 0.2% saponin or 0.2% digitonin) and incubate the suspension for 15 Min at 4°C. Wash the suspension in PBS with 0.2% Tween 20 (appr. 5 Min at 400g).

5) Block:

Block unspecific binding-sites with 5% serum or BSA, and incubate for 15-30 Min. Wash again with PBS plus 0.2% Tween 20 (appr. 5 Min at 400g).

6) Add antibodies

Add the required amount of antibodies (preliminary experiments!), and incubate the suspension in the dark for 15-45 Min at 4°C. Wash the cells with PBS containing 0.2% Tween 20 (for appr. 5 Min at 400g).

7) Resuspend the cells

Resuspend the cells in 300 to 500 µl cold PBS (or PBS with 10% FCS and 1% sodiumazide). Store the cells at 4°C until the measurements (up to 24h). The optimal results can be achieved, if the samples are measured immediately after the labelling.

Observe: This protocol is designed to help you with working with your antibodies. However, we give no guarantee on the basis of this protocol that you will achieve the best results. Consult the product's datasheet for dilutions and additional information. For additional question we are ready to help you, just »contact us.

Hint:On antibodies-online.com you will find more than 40.000 antibodies for flow cytometry (FACS)