Author: Zania Stamataki
Cell labelling for flow cytometry
Cell Surface labelling
All procedures/Abs in blocking buffer: 1%BSA/0.01%NaN3/PBS/(2% normal goat serum optional). NB Sodium Azide is very toxic and will render the cells metabolically inactive
– therefore it is not suitable for assays requiring receptor trafficking in live cells.
1. Harvest and count cells, allowing for 3x10^5 – 1x10^6 cells per stain depending on
the required number of acquisition events for FACS.
2. Centrifuge cells (1500rpm, 3min) and re-suspend in blocking buffer at 3-10x10^6
cells/ml.
3. Place 50-100ul/well of a 96-w U-bottom plate. Block for 20min (on ice if lymphocytes).
4. Wash cells and add primary (non-conjugated) Abs (e.g. anti- DC-SIGN). Incubate for
0.5-1hr @ RT or on ice according to cell type.
5. Wash cells x 2 with 150ul blocking buffer/well.
6. Add secondary (conjugated) Abs (e.g. anti-mouse IgG2a-488), incubate for 0.5-1hr.
7. Wash cells x 2 with 150ul blocking buffer/well.
8. To fix before analysis, re-suspend in 100ul 1%PFA/PBS, and incubate for 5min.
9. Wash cells x 1 with 150ul blocking buffer/well.
10. Re-suspend cells in 100ul PBS and transfer to FACS tube.
Intracellular labelling
Perm/block buffer: 1%BSA/0.1%Saponin/PBS/(2% normal goat serum - optional).
1. Harvest and count cells, allowing for 3x10^5 – 1x10^6 cells per stain depending on
the required number of acquisition events for FACS.
2. Place 50-100ul/well of a 96-w U-bottom plate.
3. Add 100ul 1%PFA/PBS, re-suspend and incubate on ice for 5min.
4. Wash cells and resuspend in perm/block buffer, incubate at RT for 20min.
5. Wash cells and add primary (non-conjugated) Abs (e.g. anti- DC-SIGN). Incubate for
0.5-1hr at RT.
6. Wash cells x 2 with 150ul perm/block buffer/well.
7. Add secondary (conjugated) Abs (e.g. anti-mouse IgG2a-488), incubate for 0.5-1hr at
RT.
8. Wash cells x 3 with 150ul perm/block buffer/well.
9. Re-suspend cells in 100ul PBS and transfer to FACS tube.
In case both cell surface and intracellular labelling is required, follow the protocol for the
cell surface antigens first and stain with the primary antibodies. Then fix and permeabilise
the cells, stain for intracellular antigens as described and add the fluorochrome
conjugated reagents for both cell surface and total antigens together in the end.
Needed for every cell type used:
1. Unstained cells treated as the rest of the cells to set SSC/FSC settings.
2. Single stains to set compensation levels.
3. Isotype control stains for each of the isotypes used as primary antibodies.