Incucyte® Live-Cell Imaging
and Analysis Best Practices
Technical Note
This technical note covers thefollowing suggestions based onbest practices for:
 Cell Seeding Well Volume
 Media Formulation andExchanges
 Objects Within Light Path
 Empty Wells
 Vessel Positioning

Overview
The Incucyte® Live-Cell Analysis System is an automated image acquisition and analysis instrument that operates, captures,
and analyzes images of cells from within a standard tissue incubator. Cells used in kinetic multi-well assays in theIncucyte® Live-Cell Analysis System typically run for extended time periods. As a result, cells must be maintained in a healthy
state, with enough nutrients for the duration of the experiment, as well as proper assay set-up to ensure conditions for highquality, reproducible image acquisition of cells over time. These factors, which make for best assay practices when studying
cell biology in a long-term kinetic fashion, are not necessarily required for traditional end-point/single time-point analysis.
However, all parameters should be optimized in order to achieve superior assay performance.

Best Practices – Cell Seeding

Best Practices – Well Volume Plate Type

Cell Density: Frequently dictated by the functional
assay being performed, cell density is a key player inassay performance. As cells approach 100% confluence,contact inhibition will begin to slow growth and can
impact cell health. For most assays, a low density range
for both adherent (1,000-5,000 cells) and suspension
cells (5,000-30,000 cells) should be considered during
assay optimization. Some assays are density driven, such asIncucyte® Scratch Wound Migration and Invasion Assays,
and require confluence to be closer to 100%. In general, it iscrucial to optimize the cell density for each assay type as thiswill ensure high quality images can be acquired, processed,and analyzed to achieve reproducible, robust data.

The volume of liquid within a well is critical to consider, notonly for maintaining cell health, but also for eliminating
deformation in image acquisition. Too low of a well volume
will cause poor image quality, even with Incucyte’s ®
algorithm to correct for deformation in transmitted light.
In general, all assays will benefit from using more volume inthe wells, when applicable. The Incucyte® Live-Cell Analysis
System is able to support over several hundred vessel types
based on the application or software module; however,
we have highlighted below commonly used microplates
and their recommendations for assay set up and image
acquisition.

Cell Settling: Uneven cell distribution can have a significant
impact on the quality of data obtained in a live-cell assay due
to the potential for variance both in and between wells. Cells
accumulating at well edges or concentrating in the centerof a well are commonly observed phenomena, in particularin outer wells, leading to some researchers to not use thesewells due to concerns about data reliability. Cell distribution
within a well is strongly determined by convection currents
which circulate within a well during warming of culture
medium. If cells are present in suspension at this moment of
fluid flow, they are more likely to be moved by these currents.
To eliminate or reduce the impact of this effect, it is strongly
recommended to allow cells to settle on a flat surface atambient temperature following cell seeding for at least 20
minutes (45-60 for suspension cell types in assays such as
chemotaxis). This settling period allows cells to sediment tothe base of the well and interact with tissue culture plastic orsurface coating, limiting the movement of cells and creating
a more homogeneous cell distribution.
Surface Coating: Surface coatings have increased
importance for long term assays because it can impact thegrowth and health of cultures over time. If these conditions
are not properly optimized, it can impact the performanceof the assay. For some functional assays, like proliferation
studies, non-adherent (suspension) cell lines should beadhered to the vessel surface using a coating like poly-Lornithine (PLO). Adherent cell lines typically do not requirea coating. In more complex functional assays, like neuronal
assays, stem cell assays and chemotaxis studies, a surface
coating material might be required for both adherent andnon-adherent cells alike. Some examples of coating material
include poly-D-lysine, poly-L-orthinine, laminin, fibronectin,
or gelatin. Our assay specific protocols provide more details
around surface coating optimization suggestions per 2D and3D applications.

Plate Type
 96-well plates: We typically recommend a range of 50200 µL medium per well. The lower end of that range
can be used for short-term assays (e.g. 24 hours) while
the higher end should be used for longer assays or whenmedia exchanges are required. The standard well volume
we use in house is 100 µL.
 384-well plates: We typically recommend a range of 4080 µL medium per well. The lower end of that range can beused for short-term assays (e.g. 24 hours) while the higher
end should be used for long-term assays (> 48 hours)
or when media exchanges are required. A standard wellvolume we use in house is 60 µL.
 Flat vs. round bottom: Round bottom plates can holdmore medium than flat bottom plates and are typicallyused in Incucyte® Single Spheroid Assays. Note that forlong-term assays, more volume will also aid with partial
media exchanges.

Assay Set Up
 Assay Type: For some applications, shorter assays canbenefit from lower volumes per well so long as users do
not go too low, where image quality will be impacted.
For some of our functional assays, like spheroid andchemotaxis, volume amounts will be noted in the protocol
for best assay practices. We typically do not recommend
using lower than 40-50 µL of medium.
 Assay Duration: Users should take into consideration thelength of the assay to support cell health. Longer assays orassay where users will have to perform media exchanges
should have a higher volume of media present duringexperiments.
 Scan Frequency: Scheduling scans at a higher frequency
can lead to a greater amount of heat generation due

to the mechanics of the Incucyte® system. This may inturn increase chances of evaporation. Therefore, with avery active scan schedule, more volume in the wells isrecommended.

Best Practices – Assay MediaMedia Formulation: In traditional end-point assays,
researchers typically optimize a functional assay with theirpreferred cell lines and preferred cell line specific medium.
That said, there are cases where users might want totransition cells to an alternative media for live-cell imaging.
For example, riboflavin can cause high levels of background
fluorescence when imaging in the green channel. Someassays would be better served by using a low-riboflavinmedia in place of media with a higher riboflavin content
for imaging reasons. Additionally, cell line properties maychange when grown in different media formulations, andthese changes can be critical when running longer term
assays; for example, cells may proliferate and/or migratedifferently in different types of media. For this reason, it
is recommended to optimize media formulations for thebest cell line performances, in particular for assays such aschemotaxis. When preparing medium, it is recommended
to filter additional components, e.g. FCS, with a 0.22 µmfilter (such as the Sartorius Minisart®).
Media Exchanges: Re-feeding cells is a typical workflow incell culture maintenance, where cell medium is exchanged
every 2-3 days. This practice should be continued in longterm kinetic assays that go beyond 72 hours to maintainessential nutrients for cell health. However, there are assays
were re-feeds will not be possible, such as toxicity assays
and phagocytosis bioparticle assays. Assays where the cells
begin to lose membrane integrity will also not be served
well by a media change. Finally, assays using suspension
cells, even when attached to a surface, will also be affectedby media exchanges. It is important to understand thenature of your assay and its duration to account for any
media changes that may be required. For some sensitive
cells, such as neurons, we recommend doing a half media
change rather than a full to preserve the samples.

Best Practices – Objects Within Light PathAs the Incucyte® Live-Cell Analysis System is an imagingsystem utilizing transmitted light, any object within the lightpath can interfere with it and generate artifacts within theimages. As such, we have a number of recommendations toprevent these artifacts occurring:
Bubbles: Bubbles are high contrast objects which caninterfere with the light path of an imaging system andimpact data analysis in the Incucyte®. We recommend theuse of reverse pipetting to limit bubble formation when
preparing vessels for imaging in the Incucyte®. Shouldbubbles still be present, we suggest removing the innerstraw from a standard laboratory wash bottle, adding a little
70% ethanol to the bottle, and blowing the vapor over thewells to break the bubbles. The ethanol is sterile so will notcontaminate a sample and will break the surface tension of
the bubbles.
Condensation: A standard effect of transferring a tissue
culture vessel from the culture hood to a warm, humid
incubator is condensation on the plastic of the vessel. Thiscan lead to series of water droplets which can impact thetransmission of light through the vessel. To prevent thisfrom occurring, we recommend allowing the vessel to warm
for at least 20 minutes to allow condensation to evaporate
before imaging commences. An alternative solution toallow immediate imaging is to pre-warm an additional
vessel whilst preparing the sample vessel. The lids are thenswitched so that the warmed lid is used in the Incucyte®
assay allowing imaging to commence immediately.

Fingerprints, Markings, Scratches
 Vessels should be handled with gloved hands on thesides of the vessel, not on the top and the bottom due tothe potential for residue to be left on the vessel.
 All labelling should be made in areas where imaging will
not occur, for example, the sides of the plate.
 Vessels with a large number of scratches in the plastic
should be avoided. Scratches are high contrast objects
that can interfere with both focusing and masking. As
such, we recommend that if batches of plates contain alarge number of scratches, then they should not be used
for live-cell imaging.
 We suggest any media droplets on the lid of the vessel
are carefully wiped away with a lint-free cloth.

Best Practices – Empty Wells
The Incucyte® Live-Cell Analysis System utilizes animage based autofocusing approach to determine themost appropriate z-plane in which to obtain an image byassessing contrast levels throughout the z-axis. To optimize
this process, information from preceding wells is used toinform the system as to the most likely focal plane in which
to image in subsequent wells. If however there are no cells
present within the well, there is the possibility that theIncucyte® will struggle to find a focal plane or find contrast
in a focal plane away from that of the cells in downstream
wells leading to an out-of-focus image. To avoid thispossibility, and to speed up scan times, users can simply
select a scan pattern which corresponds only to the wells
which contain cells when setting up a vessel in the Add
Vessel guided interface within the software.

Best Practices – Vessel Positioning

When loading vessels into the Incucyte® Live-Cell Analysis
System, we recommend removing the metal tray whichholds the vessel from the Incucyte® gantry and placing iton a flat surface. This allows correct positioning of vessels
in all trays and downwards pressure to be applied whenusing multi-well plates, with well A1 positioned in the top left
corner. This is important for fixing a vessel in position due
to the ball bearings in this tray type which hold the vessel inlocation. Inserting a vessel whilst in situ can lead to vessels
not being seated correctly within the tray. An exception tothis is the situation where a tray is already occupied with anexisting vessel. In this circumstance we would recommend
giving resistance to the underside of the tray, preventing
downward pressure being applied to the gantry arm andallowing the vessel to be seated correctly without impacting
an existing experiment.

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Specifications subject to change without notice. © 2021. All rights reserved. Incucyte and all names of Sartorius products are registered trademarks and the property of Sartorius AG and/or
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