Miltenyi "MicroBead" Artifacts Detected in T cells after 72 hours of Incubation
Miltenyi nanoparticles are retained by T cells after 72 hr incubation
What you’re about to see:
- Confocal, iso-surface, and TEM images reveal Miltenyi nanoparticles internalizing in human CD3+ T cells.
- Miltenyi nanoparticles accumulating in human CD3+ T cell vesicles.
What this means:
After 72 hours (see new TEM's below), Miltenyi nanoparticles are not biodegraded. In contrast, Dynabeads are released and removed from the cells.
Notes: All video imagery is actual footage and unaltered.
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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.
See more data
- Ex vivo isolation protocols differentially affect the phenotype of human CD4+ T cells, (abstract) Bernard et al 2002
- Cell response to dextran-derivatised iron oxide nanoparticles post internalisation,(abstract) Berry et al 2004
- Nanotoxicity of iron oxide nanoparticle internalization in growing neurons, (abstract) Pisanic et al 2007
More TEM images of Miltenyi nanoparticles before and after 24, 48, and 72 hr incubation
24 hrs post isolation: Epon sections of CD3 isolated cells incubated at 37°C, showing large multivesicular bodies containing Miltenyi nanoparticles (black specs) at high magnification.
48 hrs post isolation: Epon sections of CD3 isolated cells incubated for at 37°C. High magnification of Miltenyi nanoparticles containing electron-dense compartments.
72 hrs post isolation: Epon sections of CD3 cells incubated at 37°C. Miltenyi nanoparticles in different intracellular compartments in two neighboring CD3-positive cells.
Avoid Artifacts - Use Dynabeads®
Miltenyi vs. Dynabeads® Comparison Table
|None||Uniformity||Spherical, 1-3% variability|
|Yes||Internalized into T-Cells?||No|
|Yes||Retained in T-Cells |
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