Direct staining of cell-surface markers with fuorochrome-conjugated antibodies

Direct staining of cell-surface markers (e.g. CD antigens) is being carried out with fluorochrome-conjugated antibodies. We suggest the following steps:

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

2) Add antibody:

First of all, add the amount of 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). In case you intend a double-staining you need to add the antibody for this concomitantly with the first antibody.

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 appr. 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. Make sure the cells are re-suspended thoroughly before being measured. To do so, vortex each tube before putting them into the FACS.

Observe: 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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