Application Note 25

Serial Connection of Luer-Slides for Flow Experiments
The ibidi Pump System is specially designed
for long-term experiments of cells underperfusion. The standard setup connects one
slide to one Perfusion Set. In some cases,
however, it might be beneficial to increase thenumber of slides (i.e., cells), for example, whenplanning numerous stainings or if the cellnumber is not sufficient for the subsequent
applications (e.g., Western Blot, see
Application Note 38: Western Blot Analysis
with Cell Samples Grown in Channel-µ-Slides(PDF).
This Application Note is a step-by-step protocol showing how to create a serial connection
between two µ-Slide I 0.6 Luer channels. The protocol only specifies the procedure for two slides
but can be applied to a larger number of slides correspondingly.
Important Notes: Serially connected slides experience the very same flow rate and hence
the very same shear stress.
Since this is not the case in parallelly connected slides, we absolutely recommend connecting
the slides in series, but not in parallel!
We recommend connecting not more than 4–5 µ-Slides in the described way.
Related Documents
Application Note 3: Cell Culture in ibidi Channel Slides (PDF)
Application Note 13: Endothelial Cells Under Perfusion (PDF)
Application Note 31: Serial Connection of µ-Slide VI 0.4 Channels for Flow Experiments (PDF)
Instructions µ-Slide I Luer Family (PDF)
Instructions ibidi Pump System (PDF)

Material••
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2 µ-Slides I 0.6 Luer, ibiTreat (80186, ibidi)
Endothelial cells (e.g., HUVECs)
Endothelial Cell Growth Medium (e.g., PromoCell)µ-Slide Rack (80003, ibidi)
ibidi Pump System (10902, ibidi, please see the ibidi Pump System Instructions for details)
Perfusion Set (e.g., RED, 15 cm, ID 1.6 mm, 10962, ibidi)
Hose Clip (part of the ibidi Pump System)
Serial Connector (10830, ibidi; for alternative assembly, see page 2)
Sterile syringe (1 ml) with simple Luer adapter (various suppliers)
Standard cell culture equipment (sterile working bench, cell detachment kit, culture flasks,
pipets, tips, etc.)____________________________________________________________________________________
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Alternatively, assemble the Serial Connector using the following parts:••

Silicone Tubing 1.6 mm ID (10842, ibidi)
Luer Connector Male (10824, ibidi)
Shorten the tubing to a length of 6 cm. Thenplug one plastic connector into each end of
the tubing. This results in a connector tubing
with two free male Luer adapters at the ends
to connect channels between slides. Finally,sterilize the connector with ethanol or byautoclaving.

Figure 1: Example of an assembled SerialConnector.

1. Preparations
Important Note: Equilibrate all material, such as the µ-Slides, the cell culture medium, theSerial Connectors, and the Perfusion Set overnight in the incubator at 37°C and 5% CO2.
This step is essential to prevent air bubbles from emerging over time during the experiment.

1.1. Cell Seeding
Perform all the following protocol steps under sterile working conditions!
First, unpack the µ-Slides and place them on a µ-Slide Rack or petri dish to prevent the slides
from cooling down while working on the cold metal surface of the laminar flow hood. Next, cover
the Luer adapters with the caps that come with the µ-Slide, while preparing the cell suspension.
• Prepare a cell suspension of 1.67 x 106 cells/ml to achieve a final cell number of 1 x 105
cells/cm2 or 2.5 x 105 cells/µ-Slide.
• Remove all caps from the reservoirs.
• Fill the channels with 150 µl cell suspension. Only the inside of the channel is filled (Fig. 2A).
• Put the caps on the Luer adapters and incubate the slides at 37°C and 5% CO2 forapproximately one hour, allowing the cells to attach.
• After cell attachment, fill each reservoir with 60 µl cell-free medium and close them with the caps.
• The cells/slides are now prepared for serial connection. Depending on cell growth andmetabolism, the slides can be stored in the incubator for up to 24 h. For most experiments,
the cells should form an optically confluent monolayer.
For a detailed description of the cell seeding procedure, please refer to the µ-Slide I Luer
Instructions, Application Note 3, "Cell Culture in ibidi Channel Slides”, and Application Note 13,“Endothelial Cell Culture Under Perfusion".

1.2. ibidi Pump Setup
Set up the ibidi Pump as described in the ibidi Pump System Instructions and Application Note
13, “Endothelial Cell Culture Under Perfusion”. Fill the Perfusion Set with its total working volume,
adding media to each reservoir to create a 5 ml equilibrated medium each reservoir. Then remove
the air bubbles by running the ibidi Pump with a middle range pressure (around 50 mbar). Whilerunning the program, gently tap on the tubes and the adapters with your fingers to remove the airbubbles. The level of both reservoirs should be re-equilibrated to 5 ml after all bubbles are
removed from the tubes.
Important Note: It is crucial to remove all air bubbles before connecting the slides to the Fluidic
Unit! Any gas remaining in the system can influence the flow rate and, in the worst case, stop
the flow or flush the cells away.
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2. Connecting the µ-Slides I Luer 0.6
After cell seeding and attachment, follow these steps to connect 2 µ-Slides I Luer 0.6 serially.
Perform these steps as fast as possible to reduce cellular stress caused by temperaturefluctuations or too much agitation, which would lead to cell detachment.
Use caution when connecting the male Luer adapter to the female Luer port to help reduce
trapping air bubbles within the channel.
We recommend placing the µ-Slides on a µ-Slide Rack or petri dish to prevent the slide and thecells from cooling down while working on the cold metal surface of the laminar flow hood.
•Put the µ-Slide Rack, the syringe, the equilibrated
medium, and the equilibrated Serial Connector into
the laminar flow hood. Place the µ-Slides containing
the cells on the µ-Slide Rack (Fig. 2A).

•Fill all µ-Slide reservoirs with cell-free medium until
the top. There should be a small hump of liquid (Fig.
2B).

•Plug one male Luer adapter of the Serial Connector
into one of the female Luer adapters of one µ-Slide(Fig. 2C). Twist the adapter while tightly pressing it
into the Luer port. Wipe off any overspilled medium
using a paper towel.

•The Serial Connector is now connected to the firstµ-Slide (Fig 2D).

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Fill the syringe with at least 1 ml of prewarmed
medium. Reverse the syringe and push out thesurplus air. There should be a small hump of medium(Fig. 3A).

•Connect the syringe to the free Luer adapter of thefirst µ-Slide. Make sure no air bubbles are trapped
inside (Fig. 3B). Wipe away any spillover with a paper
towel.

•Very carefully, push the medium through the channelof the µ-Slide (Fig. 3B) until the Serial Connector
tubing is filled entirely with medium and you see asmall hump of liquid (Fig 3C).

•Plug the male Luer adapter of the Serial Connector
into the adjacent female Luer adapter of the secondµ-Slide (Fig. 3D–E).

•The slide is now ready for connection to the Perfusion
Set (Fig 3F).

•In case you want to connect more than two slides,
keep the syringe connected to the first slide. Place asecond connector tubing onto the free adapter of thesecond slide, push the medium through using thesyringe, and continue as described.

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3. Connection to the Perfusion Set
For detailed instructions for connecting the slides to the Perfusion Set, please refer to Application
Note 13 “Endothelial Cell Culture Under Perfusion”, section 6 “Connecting the μ-Slide I 0.6 Luer tothe Perfusion Set.
Shortly, perform the following steps:•

Stop the flow of the ibidi Pump using thePumpControl software. Next, disconnect theFluidic Unit (with the mounted Perfusion Set, filledwith medium) from the ibidi Pump and place it inthe laminar flow hood close to the seriallyconnected µ-Slides I 0.6 Luer. Pinch off thePerfusion Set tubing using the white plastic hose
clip (Fig. 4A).

•Pull out one male Luer adapter from the middle
connector by holding it upwards (Fig. 4B). Avoid
trapping air bubbles inside.

•Tightly connect this male Luer adapter to the freefemale Luer port of the µ-Slide (Fig. 4C).

•

The Fluidic unit is now connected to one µ-Slide(Fig. 4D).

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Slowly remove the syringe from the otherµ-Slide (Fig. 4E).

•Check the Luer port for air bubbles (Fig. 4F) and, if
necessary, remove them with a syringe or pipet.

•Disconnect the second male Luer adapter fromthe middle connector by holding it upwards (Fig.
4B) and connect it to the remaining female Luer
port on the µ-Slide (Fig. 4G).

•The serially connected µ-Slides I 0.6 Luer are now
connected to the Fluidic Unit. Wipe off any
excessive medium with a paper towel andremove the white plastic clamp (Fig. 4H).

Important Note: We recommend checking thecells under the microscope before starting theperfusion experiment.
It is crucial that the cell layer is confluent, and thecells are well adherent when they are exposed toshear stress. If the cells become stressed by theconnecting procedure, give them a few hours torecover in the incubator before starting the flow.

•Put the whole assembly into the incubator andconnect the Fluidic Unit to the ibidi Pump System.

•Start the flow experiment.

4. Adapting the Flow Rate
The software does not provide a standardized setup of more than one µ-Slide. Therefore, you will
have to manually adapt the parameters of the ibidi Pump for the new demands.
Within the software, as a guideline, you can still select channel slide, µ-Slide VI 0.4 and yourPerfusion Set color setup. But the resulting flow rate (and hence also the shear stress) will be lower
than in the single channel application.
Start a cycle with the desired shear stress and measure the flow rate by hand (see the ibidi Pump
Instructions, page 40). Then open the recalibration dialogue and insert the calculated (given flowrate of the software) and the measured flow rate.
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