ibidi µ-Plate 96 Well 3D High-Throughput 3D Cell Culture and Angiogenesis Assays Instructions
- June 12, 2024
- ibidi
Table of Contents
ibidi µ-Plate 96 Well 3D High-Throughput 3D Cell Culture and
Angiogenesis Assays
Product Information
This product is an ibidi Polymer Coverslip made from a specific material
with optical properties. It has a refractive index of nD (589 nm) and an Abbe
number. The coverslip has a certain thickness and is treated with bitrate. It
has a shelf life of 36 months. The coverslip has specific geometry dimensions
for shipping and storage conditions.
Product Usage Instructions
Tub Formation Assays
In a tube formation assay, follow these steps:
- Prepare your gel matrix according to the manufacturer’s protocol or reference.
- Let the gel polymerize under appropriate conditions.
- Use the coverslip as soon as possible.
- If storage is needed, fill sterile water around the wells to generate a humidified environment to hinder evaporation.
- Conduct your experiment.
Seeding Cells in 2D
Attention! Please refer to the instructions on page 3 for experimental
setups regarding seeding cells in 2D.
Microscopy
Note that when using gel matrices, the optical quality and the use of high-
magnification objective lenses might be restricted.
Chemical Compatibility
This coverslip is compatible with certain chemicals and solvents. Please refer
to the table on page 4 for compatibility information.
Immersion Oil
When using oil immersion objectives with the ibidi Polymer Coverslip, only
use the specified immersion oils listed in the table on page 4. Using non-
recommended oils could damage the coverslip and harm objectives and microscope
components.
Ordering Information
This product can be ordered using the following catalog numbers:
- 81506 – Description
- 81501 – Description
- 81507 – Description
- 89646 – For research use only!
Instructions μ-Plate 96 Well 3D (formerly μ-Plate Angiogenesis 96 Well)
The ibidi product family is comprised of a variety of μ-Slides, μ-Dishes, and
μ-Plates 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 μ-Plate 96Well 3D
(formerly μ-Plate Angiogenesis 96 Well) is a cell culture product for tube
formation, angiogenesis assays and direct cell culture in a standard multi-
well format. Cells can be grown on or in gel matrices, e.g. MatrigelTM or
directly on the ibidi Polymer Coverslip.
Overview
This document is applicable to the following product numbers: Cat. No. Product Name 89646 μ-Plate 96Well 3D ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized, individually packed
Material
ibidi μ-Slides, μ-Dishes, and μ-Plates are made of a polymer that has the
highest optical quality. The polymer coverslip 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 intended for one-time use and are not
autoclavable, since they are only temperature-stable up to 80°C/175°F. Please
note that gas exchange between the medium and the incubator’s atmosphere
occurs partially through the polymer 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 suitable oils can be found on page 4.
Shipping and Storage
The μ-Slides, μ-Dishes, and μ-Plates are sterilized and welded in gas- permeable packaging. The shelf life under proper storage conditions (in a dry place, no direct sunlight) is listed in the following table.
Conditions
Shipping conditions Ambient Storage conditions RT (15–25°C)
Shipping and Storage
The μ-Slides, μ-Dishes, and μ-Plates are sterilized and welded in gas-
permeable packaging. The shelf life under proper storage conditions (in a dry
place, no direct sunlight) is listed in the following table.
Conditions
Shipping conditions Ambient Storage conditions RT (15–25°C)
The μ-Plate 96Well 3D meets all-important values of the ANSI/SLAS (SBS) Standards (1-2004, 2-2004, 3-2004 and 4-2004).
- Dimensions of the μ-Plate 96Well 3D in mm
- Length 127.7 ± 0.2
- Width 85.5 ± 0.2
- Height with lid 16.5 ± 0.4
- Height without lid 14.4 ± 0.4
- Well-to-well distance 9.0 ± 0.1
Instructions μ-Plate 96 Well 3D
(formerly μ-Plate Angiogenesis 96 Well)
Single Well Parameters
- Volume inner well 10 μl
- Diameter inner well 4 mm
- Depth inner well 0.8 mm
- Volume upper well 70 μl
- Diameter upper well 5 mm
- Growth area inner well 0.125 cm2
- Coating area using 10 μl 0.23 cm2
- Well clearance 0.82 mm
- Focal offset 1 mm
Surface
The μ-Plate 96Well 3D is available with the bitrate surface. The bitrate surface is a physical treatment and is optimized for the adhesion of most cell types. Many cell lines as well as primary cells were tested for good cell growth. A specific coating of the μ-Plate 96Well 3D can be done by yourself following the procedure in the section ”Coating”.
Coating
In tube formation assays the μ-Plate 96Well 3D is coated with a 0.8 mm thick
layer of gel matrix.
- Prepare your gel matrix according to the manufacturer’s protocol or reference.
- Fill the inner well with 10 μl liquid gel. Avoid air bubbles.
- Let the gel polymerize under appropriate conditions.
- Use as soon as possible.
- If storage is needed fill sterile water around the wells to generate a humidified environment to hinder evaporation.
Non-gel-based coatings are also possible. Please use 10 μl coating solution and calculate the area to be coated of 0.23 cm2 per well. Further information about coatings is provided in Application Note 08 ”Cell culture coating”.
Tube Formation Assays
In a tube formation assay cells are seeded on top of the polymerized gel
matrix:
- Trypsinize and count cells as usual. Dilute the cell suspension to the desired concentration. Depending on your cell type, we recommend 1–3 ×105 cells/ml.
- Apply 70 μl of the cell suspension into the upper well. Do not touch the gel matrix with the pipet tip.
- Cover the μ-Plate 96Well 3D with the supplied lid. Incubate at 37°C and 5% CO2 as usual.
- Conduct your experiment.
- Depending on the cell type, medium exchange is necessary every 1–2 days. Carefully aspirate the old medium and replace it by 70 μl fresh medium. For a detailed protocol please refer to Application Note 19 ”Tube Formation” and Application Note 5 ”Tube Formation in μ-Plate 96Well 3D”. Further information about the optimization of experimental parameters and data analysis is provided in Application Note 27 ”Tube Formation – Data Analysis”.
Tip
Air bubbles in the gel can be reduced by equilibrating the μ-Plate 96Well 3D
before usage inside the incubator overnight. In case bent gel surfaces are
created, increase or decrease the amount of gel used, until you get flat and
even gels.
Tip
For less evaporation, the reservoirs at the edges can be filled with sterile
water or agarose. Add agarose to water or buffer solution (e.g. 0.1 g to 10 ml
water). Melt agarose solution using a microwave or boiling water bath and
allow the solution to cool to ∼50°C.
Tip
You can stack the μ-Plate 96Well 3D to save space in your incubator. This will
not affect cell growth. We recommend making batches with up to 6 plates, due
to stability reasons.
Seeding Cells in 2D
You can also use the μ-Plate 96Well 3D for a standard 2D cell culture without
a gel matrix.
- Trypsinize and count cells as usual. Dilute the cell suspension to the desired concentration. Depending on your cell type, the application of a 1.8– 4.3 × 105 cells/ml suspension should result in a confluent layer within 2–3 days.
- Apply 10 μl cell suspension into each well of the μ- Plate 96Well 3D. Avoid shaking as this will result in an inhomogeneous distribution of the cells.
- Cover the slide with the supplied lid. Incubate at 37°C and 5% CO2 as usual.
- After cell attachment, add 70 μl cell-free medium to fill the upper well.
Attention!
Avoid evaporation during seeding and cell culture in the incubator! We
recommend placing the μ-Plate 96Well 3D in an extra humidity chamber (e.g. a
Petri Dish with wetted paper). Undemanding cells can be left in their seeding
medium for up to three days and grow to confluence there. However, the best
results might be achieved when the medium is changed every 1–2 days. Carefully
aspirate the old medium and replace it by 80 μl fresh medium per well.
Experimental Setups
Alternatively, the μ-Plate 96Well 3D can be used for the following assays:
- Fill the inner well with cells suspended inside a gel matrix. After gel polymerization, add 70 μl cell-free medium to fill the upper well.
- Sandwich Cell Culture: Fill the inner well with a gel matrix. Seed cells on top of the polymerized gel and imbed the cells with 70 μl gel in the upper well.
- Fill the inner well with a low volume of the gel matrix, e.g. 8 μl. Seed cells, spheroids or tissue pieces on top of the polymerized gel. If necessary gently shake the slide to make the cells slide into the center of the well.
- Fill the inner well with fibroblasts suspended inside a gel matrix. Seed cells on top of the polymerized gel. Overlay the cell layer with medium and incubate for invasion of the cells into the gel matrix.
Microscopy
To analyze your cells, no special preparations are necessary. Cells can be
directly observed live or fixed, preferably on an inverted microscope. The
bottom cannot be removed. For optimal results in fluorescence microscopy and
storage of fixed and stained samples, ibid provides mounting media (50001 and
50011) optimized for μ-Dishes, μ-Slides, and μ-Plates.
Note:
When gel matrices are used the optical quality and the use of high-
magnification objective lenses might be restricted.
Chemical Compatibility
The following table provides some basic information on the chemical and
solvent compatibility of the μ-Plate 96Well 3D. For a full list of compatible
solvents and more information on chemical compatibility, please visit the FAQ
section on ibidi.com.
Chemical / Solvent Compatibility
- Methanol yes
- Ethanol yes
- Formaldehyde yes
- Acetone yes, without lid
- Mineral oil no
- Silicone oil yes
- Immersion oil See Immersion Oil on page 4.
Immersion Oil
When using oil immersion objectives with the ibidi Polymer Coverslip, use only
the immersion oils specified in the table below. The use of any non-
recommended oil could damage the ibidi Polymer Coverslip. The resulting
leakage may harm
objectives and microscope components. All immersion oils that are not listed
in the table below should be considered as non-compatible.
- Company Product Ordering No. Lot Number Test Date
- ibidi ibidi Immersion Oil 50101 16-12-27 01/2017
- Cargille Type A 16482 100592 01/2017
- Cargille Type HF 16245 92192 01/2017
- Carl Roth Immersion oil X899.1 414220338 01/2017
- Leica Immersion Liquid 11513859 n.a. 03/2011
- Nikon Immersion Oil F2 30cc MXA22192 n.a. 01/2020
- Nikon Silicone Immersion Oil 30cc MXA22179 20191101 01/2020
- Olympus Silicone Immersion Oil SIL300CS-30CC N4190800 01/2017
- Zeiss Immersol 518 F 444960 160706 01/2017
- Zeiss Immersol W 2010 444969 101122 04/2012
Ordering Information
Our well-in-a-well family is available on different surfaces and in different
formats. Please see the table below for choosing your μ-Slide or μ-Plate,
respectively.
- μ-Slide 15Well 3D
- Cat. No. Description
- 81506 μ-Slide 15 Well 3D ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized, individually packed
- 81501 μ-Slide 15 Well 3D Uncoated: #1.5 polymer coverslip, hydrophobic, sterilized, individually packed
- 81507 μ-Slide 15Well 3D Glass Bottom: #1.5H (170 μm ±5 μm) D 263MSchott glass, sterilized, individually packed
- μ-Plate 96Well 3D
- Cat. No. Description
- 89646 μ-Plate 96Well 3D ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized, individually packed
For research use only!
Further information can be found at ibidi.com. For
questions and suggestions please contact us by e-mail
[email protected] or by telephone
+49 (0)89/520 4617 0. © ibidi GmbH, Lochhamer Schlag 11, 82166 Gr¨afelfing,
Germany.