ibidi m–Slide VI 0.4 Series User manual

Instructions µ–Slide VI 0.4
The ibidi product family is comprised of a variety of µ–Slides and µ–Dishes, which have all
been designed for high–end microscopic analysis of fixed or living cells. The high optical
quality of the material is similar to that of glass, so you can perform all kinds of fluorescence
experiments with uncompromised resolution and choice of wavelength.
The convenient six channel format of the µ–Slide VI 0.4 is ideal for static cell cultivation and
the application of standard immunofluorescence protocols, like treatment, staining, and mi-
croscopy of living or fixed cells. Alternatively, the µ–Slide VI 0.4 can be connected to a pump
and enables you to observe cells under flow conditions.
Material
ibidi µ–Slides, µ–Dishes, and µ–Plates are made of a plas-
tic that has the highest optical quality. The polymer cov-
erslip on the bottom exhibits extremely low birefringence
and autofluorescence, similar to that of glass. Also, it is
not possible to detach the bottom from the upper part.
The µ–Slides, µ–Dishes, and µ–Plates are not autoclavable,
since they are only temperature–stable up to 80°C/175°F.
Please note that gas exchange between the medium and
incubator’s atmosphere occurs partially through the poly-
mer coverslip, which should not be covered.
Optical Properties ibidi Polymer Coverslip
Refractive index nD(589 nm) 1.52
Abbe number 56
Thickness No. 1.5 (180 µm)
Material polymer coverslip
Please note! The ibidi polymer coverslip is compatible
with certain types of immersion oil only. A list of suit-
able oils can be found on page 3.
Shipping and Storage
The µ–Slides, µ–Dishes and µ–Plates are sterilized and
welded in a gas-permeable packaging. The shelf life under
proper storage conditions (in a dry place, no direct sun-
light) is listed in the following table.
Conditions
Shipping conditions Ambient
Storage conditions RT (15-25°C)
Shelf Life of Different Surfaces
ibiTreat, Glass Bottom, ESS 36 months
Collagen, Poly-Lysine 18 months
Fibronectin 4 months
Geometry of the µ–Slide VI 0.4
The µ–Slide VI 0.4 provides a standard slide format accord-
ing to ISO 8037/1. The lateral adapter to adapter distance
of 9 mm (like 96 well plates) allows using multichannel
pipettes.
Dimensions
Number of Channels 6
Channel volume 30 µl
Channel length 17 mm
Channel width 3.8 mm
Channel height 0.4 mm
Adapters female Luer
Volume per reservoir 60 µl
Growth area 0.6 cm2per channel
Coating area using 30 µl 1.2 cm2per channel
Bottom matches coverslip No. 1.5
µ–Slide Surfaces
Depending on the type of cells and the special application
you are using, you will need µ–Slides with different sur-
faces. If you do not require any special adhesion molecules
for your application, the best choice will be ibiTreat, a tis-
sue culture treated surface.
The uncoated µ–Slide is manufactured from hydrophobic
plastic. For the cultivation of most cell lines, it is indis-
pensable to treat the uncoated µ–Slide with biopolymers,
which mediate cell adhesion and growth.
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Instructions µ–Slide VI 0.4
The µ–Slide VI 0.4 is also provided with a Collagen and a
Poly–L–Lysin coated surface. Such an adhesion substrate
has been shown to stimulate the adhesion and growth of
various cell lines in µ–Slides. A high quality Collagen
IV solution (Corning #356233) and Poly–L–Lysin solution
(Sigma #P4832) is used to pre-coat the slides.
Coating your µ–Slide VI 0.4
The uncoated µ–Slide must be coated to promote cell ad-
hesion. If you want to establish a certain coating to match
your needs, we recommend testing your coating proce-
dure on both uncoated and ibiTreat µ–Slides, since we
have observed that some biomolecules adhere differently
to hydrophobic and hydrophilic plastic surfaces.
• Prepare your coating solution according to the man-
ufacturer’s specifications or reference.
• Apply 30 µl and leave at room temperature for at
least 30 minutes.
• Aspirate the solution and wash with the recom-
mended protein dilution buffer.
• Optionally let dry at room temperature. Attention,
some coating proteins might degenerate when dry-
ing!
Further information about coatings is provided in Appli-
cation Note 08 Cell culture coating.
Tip:
For washing you can add the buffer into one channel
end and simultaneously aspirate it on the other side.
Seeding Cells
• Trypsinize and count cells as usual. Dilute the cell
suspension to the desired concentration. Depending
on your cell type, application of a 3–7 × 105cells/ml
suspension should result in a confluent layer within
2–3 days.
• Apply 30 µl cell suspension into the channel of the
µ–Slide. Quick dispensing helps to avoid trapped air
bubbles.
• Cover reservoirs with the supplied lid. Incubate at
37°C and 5 % CO2as usual.
• Await cell attachment in order not to flush out the
cells. Afterwards fill each reservoir with 60 µl cell–
free medium.
Tip:
The day before seeding the cells we recommend plac-
ing the cell medium and the µ–Slide into the incubator
for equilibration. This will prevent the liquid inside
the channel from emerging air bubbles over the incu-
bation time.
Trapped air bubbles can be removed from the channel
by inclining the µ–Slide and knocking at one edge.
Exchanging Medium
Aspirate both reservoirs and fill slowly 120 µl of fresh
medium into one of the reservoirs, which will replace the
channel volume by gravity flow.
Flow Application
Detailed information about flow rates, shear stress, and
shear rates is provided in Application Note 11 ”Shear
stress and shear rates” on www.ibidi.com
Suitable Tube Adapter Sets are also available (see page 4).
They consist of a tubing (20 cm) with inner diameter of
1.6 mm and adapters for the connection between the ibidi
µ–Slide (female Luer) and the tubing of the pump in use.
Please contact us for recommended perfusion setups. ibidi
provides a variety of channel slides and pump systems.
Preparation for Cell Microscopy
To analyze your cells, no special preparations are neces-
sary. Cells can be observed live, or fixed directly in the µ–
Slide on an inverted microscope. You can use any fixative
of your choice. The µ–Slide material is compatible with
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Instructions µ–Slide VI 0.4
a variety of chemicals, e.g., acetone or methanol. Further
specifications can be found at www.ibidi.com. Due to the
thin bottom of only 180 µm, high resolution microscopy is
possible.
Immersion Oil
When using oil immersion objectives, use only the im-
mersion oils specified in the table. The use of a non–
recommended oil could lead to the damage of the plastic
material and the objective.
Company Product Ordering Number
Zeiss Immersol 518 F (Zeiss) 444960
Zeiss Immersol W 2010 (Zeiss) 444969
Leica Immersion liquid (Leica) 11513859
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Instructions µ–Slide VI 0.4
Ordering Information
The µ–Slide VI 0.4 family is available with different surfaces. See table below for choosing your µ–Slide VI 0.4.
Cat. No. Description
80606 µ–Slide VI 0.4 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized
81602 µ–Slide VI 0.4 Collagen IV: #1.5 polymer coverslip, sterilized
81604 µ–Slide VI 0.4 Poly-L-Lysine: #1.5 polymer coverslip, sterilized
81601 µ–Slide VI 0.4 Uncoated: #1.5 polymer coverslip, hydrophobic, sterilized
Tube Adapter Set
Cat. No. Description
10831 Tube Adapter Set: sterilized
Selected References
G. Q. Li, G. A. Kevetter, R. B. Leonard, D. J. Prusak, T. G. Wood, and M. J. Correia. Muscarinic acetylcholine receptor subtype
expression in avian vestibular hair cells, nerve terminals and ganglion cells. Neuroscience, 2007.
A. Lorentzen, J. Bamber, A. Sadok, I. Elson-Schwab, and C. J. Marshall. An ezrin–rich, rigid uropod–like structure directs
movement of amoeboid blebbing cells. J. Cell Sci., 2011. doi: 10.1242/jcs.074849.
O. Mortusewicz, W. Roth, N. Li, M. C. Cardoso, M. Meisterernst, and H. Leonhardt. Recruitment of RNA polymerase II
cofactor PC4 to DNA damage sites. J. Cell Biol., 2008. doi: 10.1083/jcb.200808097.
M. Soyer and G. Dum´ enil. Introducing Shear Stress in the Study of Bacterial Adhesion. Journal of Visualized Experiments,
2011. doi: 10.3791/3241.
For research use only!
Further technical specifications can be found at www.ibidi.com. For questions and suggestions please contact us by e-mail [email protected]
or by telephone [phone]. All products are developed and produced in Germany.
© ibidi GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany.
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