Micro-Slides for Primary Cell Culture

March 18, 2016

IDIVAL RESEARCH INSTITUTE
MICROBIOLOGY
Principal Investigator

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Performance

Ease-of-Optimization

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Company:

Ibidi, Martinsried, Germany

Product Name:

µ-Slide 4 ibiTreat

Catalog Number:

80426

We are always on the lookout for fluorescent probes to stain cellular components in different primary cells or immortalized cell lines. Sometimes, we need multi-chambered slides, to perform parallel studies with small volumes of reagents with an excellent optical quality. Moreover, these slides should support cell growth over extended time periods. We looked for an imaging-compatible material that supports human neutrophil attachment and stability to perform our live-cell imaging experiments.

Experimental Design and Results Summary

Application

Culture of primary human neutrophils for long-term live-cell imaging.

Starting Material

Human neutrophils were isolated from peripheral blood of healthy donors using a dextran method.

Protocol Overview

Human neutrophils were isolated from peripheral blood of healthy donors using a dextran method and placed in 4-well µ-slides µ-Slide 4 ibiTreat (Ibidi, Martinsried, Germany). Cells were maintained in RPMI 1640 medium with 10% fetal bovine serum without other supplements. After 4h in culture, the medium was replaced and unattached cells were removed. The attached cells were allowed to grow for an additional 24h and the medium was preplaced with fresh medium. Time-lapse fluorescence microscopy was carried out on a Nikon Eclipse Ti-E microscope (Nikon), equipped with a PlanFluor 20× 0.6NA objective (Nikon) and a CO2 incubator (humidity-and temperature-controlled environment). One drop of NucBlue (Molecular Probes) was applied for 20 min to each well. Images were collected up to 5 hours every 1.5 min with an ORCA-R2 CCD camera (Hamamatsu) powered by Nis Elements 3.2 software. For NucBlue, a 375-390-nm excitation, 420-490-nm emission filter was used. Individual timelapse frames were imported to the open source image analysis software, ImageJ (http://rsbweb.nih.gov/ij).

Tips

Don't use more than 750 microliters culture medium per well to avoid contamination.

Results Summary

They work perfectly for our experiments. Human neutrophils generally function adherent to an extracellular matrix, so, we need a good adherence during the whole microscopy experiment. 24 hours after plating, neutrophils remain attached to the ibiTreat plastic version of these Ibidi slides, and we can begin to carry out movies after that time, without committing the viability or stability of these cells.

Features Summary

The slides are packaged separately to avoid contamination. They admit up to 750 microlitres of culture medium. Very useful also for confocal microscopy.

Additional Notes

None

Image Gallery

Summary

The Good

We particularly liked the lids which seem more secure than in other competitors. As the slides have a small area, we can also perform fixation and staining experiments (with small volume of antibodies) after recording cells by live-cell imaging.

The Bad

None

The Bottom Line

Really good product. It is affordable and if routinely established, a reliable method. We use this product now with several cell lines.

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