Protocol for immune-phenotype analysis of pDC (PBMC or whole blood) using Kit:
Staining of PBMC and whole blood to identify subset(s) of pDC is a straight forward and a standard protocol, as briefly described below.
1. Determine the number of cells required for staining which include cells for staining as well as cells for unstained control.
Note: 1x106 cells per each staining sample generally needed.
2. Add 100 ul of blood or PBMC suspension (1x106 cells in 100ul) into a labeled tube.
3. Add 10 ul of LMAX and 5ul each of all other components
Note: Single color (AF647, FITC, PE, PE-Cy7 and PerCP-Cy5.5) stained samples are recommended as compensation controls for flow cytometric analysis.
4. For PBMC, incubate for 30 min on ice. For whole blood, incubate for 20 min at room temperature (RT) in dark. After incubation, add 2 ml of RBC lysis buffer (sold separately, Cat No NBP2-29442) and continue incubation for 10 more minutes at RT .
Note: All subsequent steps are common to both PBMC and whole blood samples.
5. Add 2 ml of cold 1X staining buffer (available in product NBP2-26247). Gently vortex the tubes to mix the contents
6. Centrifuge for 10 min at 1200 RPM.
7. Aspirate/decant the supernatant being careful not to lose the cells.
8. Repeat Steps 5-7 to wash.
9. Suspend the pellet in 300 ul of 1X staining buffer and analyze by flow cytometry. Note: When not analyzing on the same day, suspend cells in 1X fixation buffer and store overnight at 4oC. The fixation buffer can be removed and the cells prepared for analysis by repeating Step 7-9 and adding 300ul of staining buffer (1x) to each tube.