Human PD-1 (programmed death-1) is a 55 kDa member of the immunoglobulin superfamily that is induced in cells undergoing apoptosis. The PD-1 protein contains an immunoreceptor tyrosine-based inhibitory motif and is expressed predominantly on activated T and B lymphocytes. PD-1 plays a key role in peripheral tolerance and autoimmune diseases and is thought to be involved in the maintenance of peripheral self-tolerance by serving as a negative regulator of immune responses. Two novel members of the B7 family have been identified as PD-1 ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC). Evidence reported to date suggests overlapping functions for these two PD-1 ligands and their constitutive expression on some normal tissues and up-regulation on activated antigen presenting cells.

Flow cytometric analysis for floating cells
We usually use Fisher tubes or equivalents as reaction tubes for all steps described below.
1) Wash the cells 3 times with washing buffer [PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3].
2) Resuspend the cells with washing buffer (5x10e6 cells/mL).
3) Add 50 µL of the cell suspension into each tube, and centrifuge at 500 x g for 1 minute at room temperature (20~25oC). Remove supernatant by careful aspiration.
4) Add 10 µL of normal goat serum containing 1 mg/mL of normal human IgG and 0.1% NaN3 to the cell pellet after tapping. Mix well and incubate for 5 minutes at room temperature.
5) Add 20 µL of PE labeled anti-PD-1 (J110). Mix well and incubate for 20 minutes at room temperature.
6) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration.
7) Resuspend the cells with 500 µL of the washing buffer and analyze by a flow cytometer.
(Positive control for Flow cytometry; transfectant)