In a Culture-Insert, how many cells do you need to seed out for cell migration experiments?
The goal is to have a confluent layer of cells in the Culture-Insert. The density of the confluent cell layer should be as consistent as possible for all of your experiments. When seeding out in a µ-Dish 35mm, we typically use the following protocol:
Prepare a cell suspension of 5 x 105 cells/ml.
Seed 70 µl / 100 µl into each well (35,000 cells per well when using the Culture-Insert 2 Well or 3 Well; 55,000 cells per well when using the Culture-Insert 4 Well).
Incubate at 37 °C for 24 hours.
Remove the Culture-Insert with sterile tweezers.
Fill the µ-Dish with 1 ml of fresh medium.
Put the µ-Dish into a stage top incubator on an inverted microscope.
Observe the cell-free gap with a 5x … 20x objective lens.
Start video microscopy (one frame for every 10 minutes), and then run for 24 hours.
How big of an area on the Culture-Insert does the 70 µl / 110 µl cell suspension cover?
The two wells of the Culture-Insert 2 Well and the three wells of the Culture-Insert 3 Well have the dimensions of 3.25 mm x 7 mm, with round corners and a radius of R=1 mm. The culture area for each well is 21.9 mm² (0.22 cm²). The outer dimensions of the Culture-Insert 2 Well are 8.4 mm x 8.4 mm, and 12.15 mm x 8.4 mm for the Culture-Insert 3 Well.
The four wells of the Culture-Insert 4 Well have the dimension of a quarter-circle with a radius of R=6.75 mm, with round corners (radius of R=1 mm). The culture area for each well is 35 mm². The outer dimension of the Culture-Insert 4 Well is a radius of R=17 mm.
Can the Culture-Inserts be used on protein-coated surfaces? Will the coating be disturbed by pulling out the Culture-Insert?
In general, the Culture-Insert can be placed on top of a coated surface, as long as the coating is dry. Note: The inserts do not stick to wet surfaces.
However, we highly recommend that you perform preliminary tests to see if removing the insert will disrupt the coating in the migration gap. You can analyze this by using a fluorophore-tagged antibody against the coating protein. If the fluorescence signal from the coating is as homogeneous and intense in the region of the migration gap as it is in the open wells, then you can assume that the coating will stay intact after the insert has been removed. Once several of these tests have been successful, then it is safe to perform the actual experiment.
After coating, how is the Culture-Insert fixed onto the dish?
The Culture-Insert is made out of a silicone material. This material is weak enough to stick onto the surface. You can simply fix it on by pressing it down. Make sure that the surface is dry.
For wound healing assays, is it important to produce damaged cells so as to mimic wounded cells on top of the gap? Or, does the Culture-Insert create a perfect gap without any wounded cells?
The Culture-Insert separates the cells from one another. It does not, however, create a wound in the classical sense (scratch). You can create a gap, and then look at the gap closure without producing a large amount of wounded cells.
With a Culture-Insert, is it possible to make wounds smaller than 500 µm?
When there aren’t any actual damaged cells, how can the Culture-Insert mimic a real wound?
In in vitro assays, it is very hard to simulate real wounds. Of course, debris from dead cells might play a role in migration, when related to wound healing. The problem is that it is not possible to create this debris in a reproducible way. ibidi’s wound healing assay using the Culture-Insert provides reproducible, non-wounded cell patches.
A bigger influence on wound healing in vivo is the interaction of different cell types, and a variety of other parameters. The ibidi wound healing assay using the Culture-Insert separates the migration speed from all of these other effects. The strength of an in vitro assay is that it can provide an objective and a reproducible experimental environment.
From time to time, the cells tend to clump at the edges of the ibidi Culture-Inserts. How can I avoid this?
We have seen this problem happen when cells were grown to a very high density in the Culture-Insert. We also noticed that with a lower cell density at the start of the experiment, this effect did not appear anymore. However, sometimes cells stick to the silicone, which indicates that the silicone is cell-compatible. This is probably what leads to the effect that you have observed. In this case, we recommend lowering the incubation time, and/or the number of cells seeded.
Sometimes it is difficult to see the confluency of cells when they are in the Culture-Inserts. Do you have any suggestions?
You are probably working with phase contrast microscopy. In small vessels, such as the Culture-Insert or 96-Well Plates, the meniscus between the air / water interface leads to a strong bending of the surface. This destroys the phase contrast effect everywhere except in the very center of the well.
One solution to this problem is to, completely and evenly, fill up the two wells of the Culture-Insert. By doing this, you create a planar surface between the medium and the air over the well. Please keep in mind, however, that you might have cross contamination between the two when doing this.
Although the material in the Culture-Inserts is autoclavable and compatible to alcohols, we do not recommend using them more than once. If you still want to try to reuse them, you need to establish a cleaning procedure using either acids or bases. ibidi does not have any experience cleaning the Culture-Inserts, and while they will probably remain sticky, debris from former experiments may influence the reproducibility.
I already have the Culture-Inserts, but I will do the assay next week. How should I store them?
Can I use the Culture-Inserts on any cover glasses?
Yes, you can use the Culture-Inserts on any cover glass. Up to now, no one has reported an incompatibility with any dry surface.
What keeps the Culture-Inserts from leaking during cell seeding?
The Culture-Insert is made out of a silicone material. The upper side is made from a rough material and the lower side is made from a very weak material, which fixes itself to a surface after the Culture-Insert is pressed down. There will not be any leaking, when the insert is properly affixed.
Have you ever had trouble getting Culture-Inserts to stick to custom-coated surfaces?
We have never had any problems getting Culture-Inserts to stick to custom-coated surfaces, as long as the surface is dry. Moist surfaces, however, will lead to leakage.
Is an application note available, which describes the coating protocol when using Culture-Inserts?
Can suspension cells (e.g., lymphoma cells) be attached to fibronectin?
That depends on the type of suspension cells you are using. You need to test the different cell types with the coating. Most cells stick to a certain coating, so you need to find out which ones will work for your experiment.
Have you tried a PORN/laminin coating with Culture-Inserts?
So far we did not test this coating with Culture-Inserts, but you can request free samples from ibidi to test this coating in your own lab.
Does the removal of the Culture-Insert disturb the coatings?
The removal of the Culture-Insert will disturb the surface coating, but in most cases, the disturbance will be small enough that there will not be any detectable effect on the migration speed of the cells. In case there is an influence on the migration speed, it will be most prominent at the beginning of the experiment. Therefore, we recommend analyzing the migration speed over the entire wound healing experiment.
Won’t the proteins be spoiled, when drying occurs on the Culture-Insert?
This depends mostly on the kind of protein being used with the Culture-Insert. People working with laminin typically prefer to keep it hydrated between coating and cell seeding. In our experience, we did not find any influence on the migration speed of cells, when the fibronectin and collagen IV were dried out.
Can 3D assays be used for non-adherent cells?
Yes, we think so. The mimicking of in vivo-like migration of non-adherent cells through interstitial tissue is a good argument for using gels in migration assays. We developed a protocol for investigating chemotactic migration of non-adherent dendritic cells in Collagen I gel.
How can you minimize cell division in migration experiments?
We have not yet established a protocol. But, from exchanges with other scientists in this field, we know that the following techniques are used:
damaging cells by using a controlled radiation
working with low serum concentrations in the cell medium
using mitotic inhibitors to decrease cell division
To a certain extent, all of these techniques will influence migration speed. We recommend eliminating any possible misinterpretations, such as observing the cross reactions with reference measurements.
Shouldn’t it be chemokinesis, instead of chemotaxis, which accelerates wound closure in a small gap, as compared to a single cells patch?
Chemokinesis is the enhancement of migratory activity, such as a reaction to the increasing concentration of a substance. It is likely that chemokinesis affects migration in most experiments. Therefore, it is important to keep the experimental conditions, which are within an experimental series, as constant as possible. In order to look for the -/- and +/+ effects of chemokinesis, you could try observing the migration of cells in larger Petri dishes (as compared to Petri dishes of a smaller size), and/or seed out cells in only one well of the Culture-Insert.
What is the advantage of impedance-based assays in comparison to using Culture-Inserts?
The electric cell-substrate impedance sensing (ECIS) assay gives you data, which avoid the image analysis part of it. If you need to collect large amounts of data, the parallelization is somewhat easier to handle. On the other hand, you will have less information on the details. For example, it would be very hard to judge whether cells move individually or in a collective way.
The ibidi Application Specialists for ECIS instruments can answer any of your more detailed questions. Please contact us at: [email protected]
Can the Culture-Inserts be used for laser microdissection (LMD)?
No, the Culture-Inserts cannot be used for laser microdissection.
Do images have to come from both the same culture dish, and the same scratch area?
Because of a 10% accuracy of the wound width, we recommend using only data from one single dish in each experiment. The data will contain more noise, such as when different positions of the wound are used at different time points. When studying the shape of a wound-healing curve, however, it would be more accurate to take the images from the same position of a wound every time.