Miltenyi "MicroBead" Artifacts Detected in T cells after 72 hours of Incubation

The Results

Miltenyi nanoparticles are retained by T cells, after 72hr incubation

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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 absorbed 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.

Dynabeads are released & removed from your cells

Positive Cell Isolation

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Negative Cell Isolation

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Did you see this YouTube video?

What immunologists say after seeing the videos


 

See comments of these results from
Dr. Denise Faustman, Associate Professor of Medicine at Harvard University


 

See comments of these results from
Jared F. Purton, PhD, Res assistant, Surh Laboratory, Scripps Research Institute.

We deeply regret the passing of Dr. Jared Purton. Please join us in supporting The Jared F. Purton Foundation to benefit charitable causes in the San Diego area.

More TEM images of Miltenyi nanoparticles before and after 24, 48 and 72hr incubation

24 Hours
 

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 Hours
 

48 hrs post isolation: Epon sections of CD3 isolated cells incubated for at 37° c. High magnification of Miltenyi nanoparticles containing electron dense compartment.

72 Hours
 

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

Miltenyi MicroBeads

 

Dynabeads FlowComp

Approx. 50nm Size 2.8µM
None Uniformity Spherical, 1-3% variability
No T-Cell Release? Yes
Yes Columns Required? No
    Yes Internalized into T-Cells? No    
    Yes Retained in T-Cells
after 72hrs?
No    

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