Please refer to chart in Suggested Companion Products section for names and sources of materials used in the following protocol
Note: The following procedures need to be performed in the hood using aseptic technique.
1. Isolate fresh human peripheral blood mononuclear cells PBMC from 60ml of fresh blood, and wash 2X with sterile 1 X PBS. Centrifuge the cells at 250 X g for 10 minutes and discard the supernatant.
2. Suspend the cells in complete medium (RPMI [Hyclone, Cat. No. SH30096]supplimented with 1% Pen/Strep, 1% L-glutamine and 10%
FBS). Count and adjust the cell concentration to 2-3 million cells/mL.
3. Label two sterile 50-mL conical tubes as "Not Stimulated" and "Stimulated with CPG". Dispense 2 million PBMC to each to
each tube (2 million cells/test)
4. Add 5µg of CPG oligodeoxynucleotide per ml of cells to the "Stimulated with CPG" tube. Cap the tube and vortex gently.
5. Incubate both tubes for 2 hours at 37°C.
6. Dilute BD FastImmune™ Brefeldin A (BFA)(Cat. No. 347688) 1 to 10 in sterile 1 X PBS.
7. Add 20µl of 1X BFA per mL to both tubes (stimulated and unstimulated). Incubate tubes at 37°C for 2 hours.
Note: Keep both CPG and BFA aliquots at -20°C.
8. Add 100µl of 20 mM EDTA per mL of cells to both tubes and incubate overnight at 4°C.
Surface and Intracellular Staining
9. Centrifuge the two tubes at 250 X g for 10 minutes. Discard the supernatants by aspiration and re-suspend cells in 25 mL of
BD Pharmigen™ Stain Buffer (Cat. No. 554656/554657).
10. Centrifuge the tubes at 250 X g for 10 minutes. Discard the supernatants and re-suspend cells in 2 mL of Stain Buffer.
11. Label 12x75mm polypropylene tubes appropriately. Add 100µl of cells to each tube.
12. Add surface staining antibodies to each tube (FITC Anti-Human Lin-1 cocktail [Cat. No. 340546], PerCP-Cy™5.5 Mouse anti-Human
CD123 [Cat. No. 558714/560904], and PE Mouse Anti-Human HLA-DR [Cat. No. 555561] or PE conjugate). Incubate the tubes at
room temperature for 30 minutes in the dark.
13. Following incubation, add 2 mL of cold Stain Buffer to each tube and centrifuge at 250 X g for 10 minutes.
Discard the supernatants by aspiration and vortex the pellets to re-suspend the cells.
14. Add 1 mL of room temperature BD Cytofix/Cytoperm™ Buffer (Cat. No. 554722) to each tube. Mix well and incubate at room
temperature in the dark for 30 minutes.
15. Add 2 mL of cold BD Perm/Wash™ Buffer (Cat. No. 554723) to each tube and centrifuge at 500 X g for 5 minutes; discard the
supernatants by aspiration and vortex the pellets to re-suspend cells.
16. Add intracellular staining antibody anti-human IFN-alpha (20µl/test) or the proper isotype control at the appropriate
volume per test to the tubes and mix well by vortexing. Bring test volume to 100µl using cold BD Perm/Wash Buffer.
Incubate tubes at room temperature for 60 minutes in the dark.
17. Add 2 mL of cold BD Perm/Wash Buffer to each tube and centrifuge at 500 X g for 5 minutes; discard supernatant
by aspiration and vortex pellet to suspend cells.
18. Add 300µl of cold BD Perm/Wash Buffer to each tube for immediate flow cytometric analysis.
Optional: Re-suspend the pellets with 200 µl of cold 1% -formaldehyde and keep the tubes at 4°C in the dark up to 24 hours before flow cytometry. If storing longer than 24 hours, we recommend washing cells in wash buffer as extended incubation with fixatives might affect fluorochromes.
Flow Cytometry and Data Analysis
Acquire at least 500,000 events (lymphocytes and monocytes).
A sequential gating strategy is required for successful data analysis:
1. Gate tightly on the lymphocytes and monocytes using scatter profiles.
2. View the Lin-1 versus HLA-DR profile of the gated lymphocytes and monocytes, and select the Lin-1-negative, HLA-DR-positive population (R2 in the figure). Be sure not to include any of the Lin-1-positive cells.
3. View the IFN- versus CD123 profile of the gated cells to detect the IFN- -positive population.
Unstimulated sample is used as a negative control could also be used to set the markers.
Open up the second gate on Lin-1- HLA-DR+(R2) and from there open up the third gate for CD123+ IFN-alpha+(R3). Acquire about 150-200 events in CD123+ IFN-alpha+ double positive quadrant (R3). See below for correct and incorrect gating examples.
The proper gating for IFN-alpha is crucial for proper analysis. Below are two examples. The first example is how we recommend gating for proper detection of IFN-alpha. The second example is the incorrect way to gate for IFN-alpha.