Uptake and intracellular accumulation of Miltenyi MicroBeads in human CD3+ T cells

Miltenyi MicroBeads: Co-localization with acidic vesicles and 24 hr retention in Human CD3+ T cells

Labeling of cells and MicroBeads

PBMC was generated from healthy blood donors by gradient centrifugation (LymphoPrep, Axis-Shield) according to the manufacture’s protocol and labeled with Vybrant Multicolor Cell-Labeling Kit (Component C, Cat. No. V22889) for 30 min at room temperature prior to washings. The tube was rolling during labeling to avoid sedimentation of the cells. In some experiments, LysoTracker Green Dye (Cat. No. L7526) was added during the incubation. CD3 Microbeads (Cat. No. 130-050-101, Miltenyi Biotec, Germany) were labeled with highly cross-adsorbed goat anti-mouse IgG (H+L) Alexa 555 (Cat. No. A21424). 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 MicroBeads

Labeled 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 captured at different intervals for various times (e.g., every other second for 15 min or every 5th second for 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.