Indirect staining of surface-markers with primary antibodies and flurochrome-conjugated secondary antibody

The indirect staining method requires two incubation-steps: first primary antibody incubation is carried out, followed by incubation with a secondary antibody. The flurochrome coupled secondary binds the primary antibody. Hence, the primary antibody becomes visible for the FACS measurement.

1) Harvest 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. Alternatively, you can use other tubes or microtiterplates (round-bottom), depending on the design of your centrifuge.

2) Add antibody:

First of all, add the amount of primary antibodies to each tube, and add the desired amount of cells (e.g. 150 µl for 150.000 cells per condition, when using 1 - 5 x 10⁶ /ml).

3) Incubate cells:

Let the cells incubate for 30 Min. in the dark. Incubation can be carried out under RT or 4°C. In this case determine the optimal conditions for your experiment previously.

4) Washing:

After incubation add 1 mL cold PBS (or PBS with 10% FCS, 1% sodiumazide) and centrifuge the suspension at approx. 400g for 5 Min. Take the supernatant and repeat the washing step twice (you might find that one washing step is sufficient in order to remove unbound antibody. Predetermine how many washing steps are needed under your experimental conditions in order to save time).

5) Add secondary antibody:

Add the right amount of flurochrome-coupled secondary antibodies to each tube (check the manufacturers instructions or perform preliminary experiments in order to determine the correct amount (recommended))..

3) Incubate cells:

Let the cells incubate for 20 to 30 Min. in the dark. Incubation can be carried out under RT or 4°C. Determine the optimal conditions in preliminary experiments.

4) Washing:

After incubation add 1 mL cold PBS (or PBS with 10% FCS, 1% sodiumazide) and centrifuge the suspension at approx. 400g for 5 Min. Take the supernatant and repeat the washing step twice (you might find that one washing step is sufficient in order to remove unbound antibody. Predetermine how many washing steps are needed under your experimental conditions in order to save time).

5) Resuspend:

Re-suspend the stained cells in 300 to 500 µL ice-cold PBS (or PBS with 10% FCS, 1% sodiumazid), and store them on ice, or in the fridge until your measurements. Optimal results are obtained if the samples are measured immediately after staining.
Please note: This protocol only serves as a suggestion. It does not entail any guarantee for optimal results. Check the data-sheet for dilutions and further details. For further questions you can »contact us.

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