Staining of Cell Surface Markers for Flow Cytometry
1. Cells for FACS staining were transferred into FACS tube and pelleted by centrifugation at 400 u g for 5mins. Tubes were inverted to discard the supernatant and were further blotted on tissue paper to remove the excess supernatant that collected near the edge of the tube after inversion.
2. The cell pellet was then disrupted by raking on tube racks. Fc Block (2.4G2) was added at 0.2g per million cells and incubated for 5 mins at 4oC to prevent non-specific binding of antibodies to Fc receptors.
3. Antibodies directly conjugated to fluorophores were added to the cells at 0.02g per million cells and incubated in the dark at 4oC for 30mins. After incubation, cells were washed twice with at least 1ml of FACS buffer to remove unbound antibodies.
4. Cells were resuspended in 350l of FACS buffer if analysis was performed within several hours or fixed with 1% PFA if analysed on a separate occasion.
5. To fix with 1% PFA, the cells were pelleted and the FACS buffer supernatant discarded. To prevent the formation of cell clumps during fixation, the cell
52 pellet was well disrupted by raking several times on tube racks and vortexed at 3000rpm while simultaneously adding 1% PFA.
Intracellular Staining for Flow Cytometry
1. Intracellular staining of antigens was performed after staining of cell surface markers. Stained cells were fixed following the protocol above and kept at 4oC for 15 mins to allow fixation to be complete.
2. Fixed cells were pelleted by spinning at 500 u g for 5 mins and the 1% PFA supernatant was discarded.
3. The cell pellet was disrupted by raking and permeablised by adding 1ml of permeablization buffer. Cells were then incubated in the dark for 10 mins at room temperature to allow permeablisation to be complete. Permeablised cells were then pelleted at 600 u g and the supernatant discarded before raking the tube to resuspend the cell pellet.
4. Antibodies directly conjugated to fluorophores were added to the cells at 0.02g per million cells and incubated in the dark at 4oC for 30mins.
5. After incubation, cells were washed twice with at least 1ml of Permeablisation buffer to remove unbound antibodies. Cells were finally resuspended in 350l of FACS buffer for analysis.
Intracellular Cytokine Staining
For intracellular staining of cytokines in cells, synthesis of cytokines was stimulated by addition of the appropriate stimulus. Cytokine secretion from the cell was blocked
53 by the addition of Brefeldin A (BD Pharmingen, USA) and Golgi stop (BD Pharmingen, USA) containing monensin into the culture medium. Both Brefeldin A and Golgi stop was added at 1l per 1ml of culture medium. After 6 hours, cells were harvested, transferred into FACS tubes before proceeding for surface staining of markers, followed by intracellular staining for cytokines.
Sorting of Dendritic Cells from the Lung
1. Lung cells were enriched for DCs using OPTIPREP as described earlier. The cells accumulating at the OPTIPREP and FCS interface were collected, counted and pelleted in sterile FACs tubes.
2. The concentration of the cells was adjusted to approximately 50 million cells/ml using MACS buffer. Where single stains were required for multicoloured cell sorting, small amount of cells were taken for single stains at this point.
3. Fc Block (2.4G2) was added at 0.2g per million cells and incubated for 5 mins at 4oC to prevent non-specific binding of antibodies to Fc receptors.
Antibodies directly conjugated to fluorophores were added to the cells at 0.02g per million cells and incubated in the dark at 4oC for 30mins.
4. After incubation, cells were washed once with 2 to 3ml of MACS buffer.
Cells were then resuspended in 2 ml of MACS buffer and passed through a 61m strainer to filter out cell clumps or any tissue pieces which may clog the cell sorter.
54 5. The filtered cells were then pelleted once more. Cells were finally
resuspended to a concentration of between 20-50 million cells/ml and DCs were sorted using the MOFLO (Beckman Coulter, USA).
6. DCs were collected in sterile FACS tubes containing complete RPMI or neat FCS.
Sorting of Dendritic Cells from the Lymph Node
The protocol for sorting DCs from the lymph node is identical to that for sorting DCs from the lung, with the exception that OPTIPREP density centrifugation is not performed. The cells obtained after digestion of the lymph node are directly stained for DC markers and are sent for cell sorting.
As DCs constitute a small minority within the lymph node of approximately 1 to 2 percent of the total cells, typically 5 to 6 lymph nodes are pooled together to obtain sufficient DC numbers for sorting. An estimated yield is about 40,000 DCs can be isolated from 5 lymph nodes at 3 days post-infection.
Tetramer Staining of Influenza-Specific CD8 T-cells
Influenza-specific CD8 T-cells were detected using the tetramer ASNENMETM H- 2Db PE (Proimmune, UK). Tetramer staining was done simultaneously with the surface staining antibodies anti-CD8 and anti-CD3 at 4oC for half an hour. The cells were washed twice in cold FACS buffer and fixed with 1% PFA before FACS analysis to prevent internalisation of tetramers by the T-cells.