Application Note 08

Coating Protocols for ibidi Labware
The growth, development, and signaling of cultured cells strongly depend on the used surface.
The ibidi labware has a thin coverslip bottom (ibidi Polymer Coverslip or Glass Coverslip, bothwith excellent optical quality). For optimized cell adhesion, protein coatings can be applied to theibidi labware family.
This Application Note provides a detailed protocol on how to coat the ibidi labware, as well as
important information about the required volumes and protein concentrations.
ibidi also offers a high-quality Collagen Type I, Rat Tail and Collagen Type I, Bovine solutions for
preparing 3D gels and surface coatings.

Table of Contents
1.

Recommended Surfaces for Different Coatings ................................................................. 22.

Recommended Protein Concentrations and Coating Volumes for ibidi Labware ................ 3

3.

Coating Protocol ................................................................................................................5

4.

Appendix ...........................................................................................................................6

4.1. Preparation of the Coating Solution: Calculating the Necessary Concentration for ibidi
Labware ............................................................................................................................6
4.2. Coating Areas and Coating Volumes of the ibidi Labware ................................................. 6____________________________________________________________________________________
Application Note 08© ibidi GmbH, Version 6.1, February 1st, 2024

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Application Note 08

1. Recommended Surfaces for Different Coatings
The ibidi labware is available with several surfaces:•ibiTreat (tissue culture-treated) surface on the ibidi Polymer Coverslip: Excellent
adhesion of adherent cells, hydrophilic surface; coating possible, but not needed for mostadherent cell types•Hydrophobic, Uncoated surface on the ibidi Polymer Coverslip: Weak adhesion ofadherent cells, hydrophobic surface; coating usually required for cell adhesion•Bioinert surface on the ibidi Polymer Coverslip: No adhesion of adherent cells or any
protein, stable long-term passivation; no coating possible•

ESS (elastically supported surface) on the ibidi Polymer Coverslip: Culture ofadherent cells on an elastic, very hydrophobic surface; coating required for cell adhesion•Glass surface on the ibidi Glass Coverslip: Adhesion of adherent cells; coating might
be required for optimal cell adhesionProtein CoatingCollagen ICollagen IVFibronectin

Poly-L-Lysine
Poly-D-Lysine

Recommended Surfaces
ibiTreat
Glass
ibiTreat
Hydrophobic, UncoatedGlass
ibiTreat
Hydrophobic, Uncoated
ESS
Glass
ibiTreat
ESS
Glass
ibiTreat
ESS
Glass

Some biomolecules adhere differently to hydrophobic and hydrophilic surfaces (e.g., ibiTreat orglass). Therefore, we recommend testing a specific coating or a protein mixture on different
surfaces before using them in an experiment.
Free samples of the ibidi labware can be ordered at: ibidi.com/freesample____________________________________________________________________________________
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Application Note 08

2. Recommended Protein Concentrations and Coating Volumes
for ibidi Labware
Here you find precalculated values of some standard coatings for the different ibidi labware
products. There is an equation for calculating the protein concentration in the Appendix.
In this Application Note, we used the following coating solutions:
Collagen I (ibidi, Rat Tail, 50202 or ibidi, Bovine, 50302)
Recommended protein amount per area: 5 µg/cm2. Dilute the Collagen Type I to the required
concentration using a) for rat tail: 17.5 mM acetic acid (~0.1% acetic acid) or b) for bovine:
0.1 M acetic acid (~0.6% acetic acid).
Collagen IV (Corning, mouse tumor, No. 356233)
Recommended protein amount per area: 1.5 µg/cm2. Dilute the Collagen Type IV to the required
concentration using 0.05 M HCl.
Fibronectin (Corning, human plasma, 354008)
Recommended protein amount per area: 1.5 µg/cm2. Dilute the Fibronectin to the required
concentration using PBS (pH 7.2) without Ca2+ and Mg2+.
Poly-L-Lysine (Sigma-Aldrich. 0.01% solution, 100 µg/ml, P4832)
Recommended protein amount per area: 2 µg/cm2. Dilute the PLL to the required concentration
using ultra-pure water.
Poly-D-Lysine (Corning, No. 354210)
Recommended protein amount per area: 5 µg/cm2. Dilute the PDL to the required concentration
using ultra-pure water.
If any coating protein products other than those specified here are being used, please follow themanufacturer's instructions for the recommended protein amount per area and preparation.
IMPORTANT NOTE:
Since adhesion proteins are biological substances, there can be quality differences between thelots of a manufacturer. Therefore, we recommend testing every lot number, especially prior tolarge scale experiments.
Recommended Coating Volumes and Protein Concentrations for ibidi Labware
Protein Concentration

501515Poly-LLysine[µg/ml]204005015152050800

100303040100µ-Dish 35 mm Quad3004513131745Glass Bottom Dish 35 mm

4005015152050

CoatingVolume [µl]µ-Dish 35 mm, low 1)

400µ-Dish 35 mm, high 1)µ-Dish 35 mm, high ESS 2)

1)
2)

Collagen I Collagen IV Fibronectin[µg/ml][µg/ml][µg/ml]Poly-DLysine[µg/ml]50

Also valid for glass bottom and Grid-50/Grid-500 versions.
For the very hydrophobic ESS surface, a higher volume and a higher protein concentration are necessary.

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Application Note 08

Protein Concentrationµ-Slide 1 Well 3)µ-Slide 2 Well 3)µ-Slide 2 Well Ph+ 3)µ-Slide 2 Well Co-Cultureµ-Slide 4 Well 3)µ-Slide 4 Well Ph+ 3)µ-Slide 8 Well 3)µ-Slide 8 Well high 3)µ-Slide 15 Well 3D 3)µ-Slide 18 Well 3)µ-Slide 18 Well - Flatµ-Slide Iµ-Slide I 0.2 Luerµ-Slide I 0.4 Luerµ-Slide I 0.6 Luerµ-Slide I 0.8 Luerµ-Slide I 0.2 Luer Glass Bottomµ-Slide I 0.4 Luer Glass Bottomµ-Slide I 0.6 Luer Glass Bottomµ-Slide I 0.8 Luer Glass Bottomµ-Slide I Luer 3Dµ-Slide III 3in1µ-Slide VI 0.4µ-Slide VI 0.5 Glass Bottomµ-Slide VI 0.1µ-Slide VI - Flatµ-Slide y-shapedµ-Slide Chemotaxis 4)µ-Slide Chemotaxis 5)µ-Slide ibiPore SiNµ-Slide III 3D Perfusionµ-Slide Spheroid Perfusionµ-Plate 24 Wellµ-Plate 96 Well 3Dµ-Plate 96 Well Round 3)µ-Plate 96 Well Square 3)µ-Plate 384 Well Glass Bottom
3 Well Chamber, removable
8 Well Chamber, removable
12 Well Chamber, removable
Culture-Insert 2 Well
Culture-Insert 3 Well
Culture-Insert 4 Well
micro-Insert 4 Well
micro-Insert 4 Well FulTrac

Coating
Volume [µl]Collagen I Collagen IV Fibronectin[µg/ml][µg/ml][µg/ml]3000
1500 / well
1500 / well
70 / minor well
700 / well
700 / well
300 / well
300 / well
10 / inner well
100 / well
30 / well
100
50
100
150
200
62.5
112.5
162.5
212.5
16 / well60
30 / channel
40 / channel
1.7 / channel
30 / channel
110
130 / chamber
6 / chamber
50 (lower channel)
130 / channel
3.5 / well1000 / well
10 / inner well
200 / well
300 / well
50 / well1100 / well
400 / well
250 / well
70 / well
70 / well
110 / well
10 / well
10 / well

24
25
38
40
30
42
35
35
125
58
40
250
500
250
200
145
420
250
175
135
100
250
250
150
1000
250
250
130
230
250
100
140
20
125
40
35
80
15
35
35
60
60
60
115
100

78
1112
912111138
171275
150
75
60
44
125
75
52
41
30
75
75
45
300
75
75
40
70
75
30
40
6
38
1212
25
5
11
11
18
18
18
35
30

78
11
12
912
11
11
38
171275
150
75
60
44
125
75
52
41
30
75
75
45
300
75
75
40
70
75
30
40
6
38
12
12
25
5
11
11
18
18
18
35
30

Poly-LLysine[µg/ml]
10
1015
17
12
1715
1550
23
17
100
200
100
80
60
170
100
70
55
40
100
100
60
400
100
100
55
90
100
40
55
950
16
15
33
615
15
25
25
25
47
40

Poly-DLysine[µg/ml]
24
25
38
40
30
42
35
35
125
58
40
250
500
250
200
145420250175135100250250150
1000
250250130230250
100
14020125403580
153535606060
115
100

Also valid for the glass bottom version.
When coating the entire chamber (observation area + 2 reservoirs).
5)
When coating the observation area only.
3)
4)

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Application Note 08

3. Coating Protocol
All steps need to be performed under sterile conditions.
1. Check the required protein concentration and coating volume, then dilute the coatingsolution to the desired concentration (see Section 2).
2. Fill the vessel (e.g., a channel or a well) with the coating solution, using the coating volumein Section 2.
Incomplete filling or large air bubbles will lead to incomplete coating.
The ibiTreat surface is easier to wet completely with therecommended volumes than the Hydrophobic, Uncoated surface.
Quick dispensing helps when filling the channel slides. Very smallchannels (channel height 0.2 mm and below) can be filled easier byusing a small volume syringe with a male Luer tip, as shown on theright.
3. Incubate at room temperature for 60 minutes.
4. Wash thoroughly.
Attention: It is necessary to remove all unbound proteins. Any remaining protein may
inhibit cell attachment.
a. Open wells: Aspirate the well volume completely. Make sure not to touch thecoated surface to keep the protein coating functional. Rinse carefully with ultrapure water or PBS. For rinsing, we recommend using 2–3x the coating volume.
b. Channels: Rinse the channel with ultra-pure water or PBS
by a continuous liquid exchange using 3–5x the channel
volume. Make sure that the channel is always filled byadding the washing solution into one channel end, followedby aspiration from the opposite end, as shown on the right.
5. Optionally, let dry at room temperature.
Attention: Some coating proteins might degenerate during drying! Coatings on theESS surface must not be dried!
6. The labware is now coated and ready-to-use. Introduce cells and medium immediately or
store it under sterile conditions and use as soon as possible.

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Application Note 08

4. Appendix
4.1. Preparation of the Coating Solution:
Concentration for ibidi Labware

Calculating

theNecessary

Before preparing a coating, you need to calculate the protein concentration and the coating
volume required for the chosen labware. This Application Note contains the precalculated valuesof some standard coatings for the different ibidi labware products.
All calculations are based on the amount of protein per area (P) [µg/cm²], which is specific forthe coating protein being used, and is recommended by the manufacturer. Importantly, optimal
conditions for cell attachment can vary and are influenced by the cell type and application. The
typical range for the protein amount per area is 1–5 µg/cm².
The calculated protein concentration (c) [µg/ml] depends on the coating area (A) [cm²] and thecoating volume (V) [µl] that is needed to cover the coating area.
The coating area, which is in direct contact with the coating solution, is defined by the usedlabware: Channels are coated on the entire inner surface; open wells are coated on the growtharea and partially on the side walls. Please see Section 4.2 for the coating areas, which are thebasis for the protein concentrations and volumes calculated in this section.
The required protein concentration for the coating of different labware is calculated as follows:
𝐜𝐜 [µg⁄ml] =𝐏𝐏 [µg⁄cm²] × 𝐀𝐀 [cm²] × 1000
𝐕𝐕 [µl]4.2. Coating Areas and Coating Volumes of the ibidi Labware
The protein concentrations for the ibidi labware in Section 2 were calculated based on the following
coating areas and coating volumes. The growth area is listed for information purposes only.

µ-Dish 35 mm, low 1)µ-Dish 35 mm, high 1)µ-Dish 35 mm, high ESS 2)µ-Dish 35 mm QuadGlass Bottom Dish 35 mmµ-Slide 1 Well 3)µ-Slide 2 Well 3)µ-Slide 2 Well Ph+ 3)µ-Slide 2 Well Co-Cultureµ-Slide 4 Well 3)µ-Slide 4 Well Ph+ 3)µ-Slide 8 Well 3)µ-Slide 8 Well high 3)µ-Slide 15 Well 3D 3)µ-Slide 18 Well 3)µ-Slide 18 Well - Flat

Growth Area [cm2]

Coating Area [cm2]

Coating Volume [µl]

3.5
3.5
3.5
0.85 / well3.1410.6
4.8 / well
4.8 / well
0.4 / minor well
2.2 / well
2.2 / well
1.0 / well
1.0 / well0.12 / well0.34 / well
0.2 / well

4.1
4.1
4.1
2.46 / well
3.7
14.6
7.5 / well
11.4 / well0.55 / minor well
4.1 / well
5.9 / well
2.2 / well
2.2 / well0.23 / well
1.15 / well0.25 / well

400
400
800
300
400
3000
1500 / well
1500 / well
70 / minor well
700 / well
700 / well
300 / well
300 / well
10 / inner well
100 / well
30 / well

Also valid for glass bottom and Grid-50/Grid-500 versions.
For the very hydrophobic ESS surface, a higher volume and a higher protein concentration are necessary.
3)
Also valid for the glass bottom version.
1)2)____________________________________________________________________________________
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Application Note 08µ-Slide Iµ-Slide I 0.2 Luerµ-Slide I 0.4 Luerµ-Slide I 0.6 Luerµ-Slide I 0.8 Luerµ-Slide I 0.2 Luer Glass Bottomµ-Slide I 0.4 Luer Glass Bottomµ-Slide I 0.6 Luer Glass Bottomµ-Slide I 0.8 Luer Glass Bottomµ-Slide I Luer 3Dµ-Slide III 3in1µ-Slide VI 0.4µ-Slide VI 0.5 Glass Bottomµ-Slide VI 0.1µ-Slide VI - Flatµ-Slide y-shapedµ-Slide Chemotaxis 4)µ-Slide Chemotaxis 5)µ-Slide ibiPore SiNµ-Slide III 3D Perfusionµ-Slide Spheroid Perfusionµ-Plate 24 Wellµ-Plate 96 Well 3Dµ-Plate 96 Well Round 3)µ-Plate 96 Well Square 3)µ-Plate 384 Well Glass Bottom3 Well Chamber, removable
8 Well Chamber, removable
12 Well Chamber, removable
Culture-Insert 2 Well
Culture-Insert 3 Well
Culture-Insert 4 Well
micro-Insert 4 Well
micro-Insert 4 Well FulTrac

Growth Area [cm2]

Coating Area [cm2]

Coating Volume [µl]

2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
0.21 / well
1.23
0.6 / channel
0.6 / channel
0.17 / channel
0.6 / channel
2.8
1.24 / chamber
0.06 / chamber
1.25 (lower channel)
0.25 / well
0.005 / well
1.9 / well0.12 / well
0.3 / well0.55 / well0.11 / well
1.66 / well
0.93 / well0.56 / well0.22 / well0.22 / well0.35 / well0.03 / well0.0012 / well

5.4
5.2
5.4
5.6
5.8
5.2
5.4
5.6
5.8
0.34 / well
3.05
1.2 / channel
1.2 / channel
0.34 / channel
1.2 / channel
5.6
3.5 / chamber
0.27 / chamber
2.7 (lower channel)
2.4 / channel
0.097 / well
4.3 / well0.23 / well1.61 / well2.35 / well0.80 / well3.37 / well2.63 / well
1.9 / well0.82 / well0.82 / well1.23 / well0.23 / well0.188 / well

10050
100
150
200
62.5
112.5
162.5
212.5
16 / well60
30 / channel
40 / channel
1.7 / channel
30 / channel
110
130 / chamber
6 / chamber
50 (lower channel)
130 / channel
3.5 / well1000 / well
10 / inner well
200 / well
300 / well
50 / well1100 / well
400 / well
250 / well
70 / well
70 / well
110 / well
10 / well
10 / well

Also valid for the glass bottom version.
When coating the entire chamber (observation area + 2 reservoirs).
5)
When coating the observation area only.
3)4)____________________________________________________________________________________
Application Note 08© ibidi GmbH, Version 6.1, February 1st, 2024

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