Labeling of cells and MicrobeadsPBMC was generated from healthy blood donors by gradient centrifugation (LymphoPrep, Axis-Shield) according to the manufacture’s protocol and labeled with Lipid Vybrant Multicolor Cell Labeling kit (Component C, cat no V22889, Invitrogen) for 30 min at room temperature prior to washings. The tube was rolling during labeling to avoid sedimentation of the cells. In some experiments, LysoTrackerTM Green (cat no L7526, Invitrogen) was added during the incubation. CD3 Microbeads (cat no 130-050-101, Miltenyi Biotec, Germany) were labeled with highly cross absorbed goat anti-mouse IgG (H+L) Alexa 555 (cat no A21424, Invitrogen). The staining of CD3 MicroBeads was specific as adding the anti-mouse IgG Alexa 555 antibody directly to PBMC did not create a signal in a fluorescent microscope.
Detection of the MicroBeadsLabeled CD3 MicroBeads were used to isolated human CD3+ T-cells according to the manufacture’s procedure (Miltenyi Biotec, Germany) using LS columns. Isolated cells were immediately analyzed on glass cover slips in an Andor Revolution Spinning Disc microscope. Frames were taken at different intervals for various times (e.g. every 2nd sec for 15 min, or every 5th sec up to 45 min). In some experiments, labeled PBMC (70% of cells expressing CD3) were seeded directly onto a glass cover slip prior to adding the labeled CD3 MicroBeads and live imaging of the cells was performed.