Mouse anti human cd45

For visualizing conjugated cells in a horizontal orientation, we attached the different types of cells to different glass surfaces that were placed one on top of the other. Two types of glass surfaces were prepared: the 8-well glass chambers and small glasses that fit the opening of the chamber well. Coverslips were incubated with 10 μg mL⁻¹ non-stimulatory antibodies (Mouse anti human CD45 (BD Pharmingen, San Diego, CA, USA, PMG555480); Mouse monoclonal IgG2a αCD11a (LFA1α) (BD Pharmingen, 555378)) overnight at 4 °C or 2 h at 37 °C. Finally, coverslips were washed with phosphate-buffered saline (PBS).
Adherent tumor cells were seeded the day prior to the experiment in a chamber well. The lymphocytes cells were dropped on an opposite small glass coated with αCD45/αCD11 antibody for 10 min in 37 °C for attachment. We then flipped the small glass on the adherent cells in the chamber well. In order to keep an appropriate space between two glasses we used 20 µm standard silica beads (47148-10, Corpuscular, Cold Spring, NY, USA). In fixed cell imaging, cells after staining and dropping, were fixed by 2.4% Paraformaldehyde (PFA) for 30 min in 37 °C and washed with PBS.