Post-sorting, the cells were collected in culture medium with 20% FCS and plated in 35 mm wells For monitoring surface expression of PF14_0325, PFD1130W or PFF0620C on transfected cell lines, cells were stained as described above.
Here we have used novel automated cell imaging methods to monitor cell surface expression and internalization of HA-tagged hGnRHRs, XGnRHRs, and chimeric receptors (hGnRHR with an added XGnRHR COOH-terminal tail) expressed by adenoviral transduction in HeLa cells.
In contrast, mHLA-DR monitors the surface expression only on monocytes.
In parallel, we monitored CR1 surface expression by flow cytometry and we assessed the levels of CR1 released in the extracellular medium by ELISA.
Further, flow cytometry to monitor surface HLA-DR expression demonstrates that transient transfection of HLA-DR induces an approximate half-log to log-fold increase in HLA-DR as compared to cells stably or transiently expressing CIITA, respectively (Figure S1).
Consequently, incubation of cells with biotinylated HB-19 and followed by purification of cell extracts using avidin-agarose provide an efficient method for monitoring surface nucleolin expression.
To examine more in depth the dynamics of CR1 oscillation, we first monitored surface CR1 expression in three patient-derived spheroid lines, which all showed a rough sinusoidal trend although with different phases.
To analyze lymphocyte populations relative to purity at different stages of the isolation procedure, cells were incubated with specific antibodies and surface expression monitored by flow cytometry.
The fluctuation of CR1 expression was not influenced by oxygen conditions (Supplementary Figure 2b) and was not determined at the transcriptional level, as CR1 RNA did not show significant differences when monitored in parallel to surface expression (Supplementary Figure 2c).
To monitor changes of adhesion molecule surface expression (β1 integrin, β7 integrin and CD62L) after epratuzumab incubation, freshly isolated PBMCs were incubated with 10 μg/mL of epratuzumab in RPMI 1640 medium (Gibco BRL, Karlsruhe, Germany) supplemented with 0.5% (w/v) BSA for 90 minutes at 37°C and 5% CO2.
