Application Note 31

Serial Connection of µ-Slide VI 0.4 Channels
for Flow Experiments
Also Applicable for Connecting Single Channels of Other Luer-Slides
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,
when planning numerous stainings or if thecell number is not sufficient for thesubsequent applications (e.g., Western Blot,
see Application Note 38: Western BlotAnalysis with Cell Samples Grown in
Channel-µ-Slides (PDF).
This application note is a step-by-step protocol explaining how to create a serial connection
between the six channels of a µ-Slide VI 0.4.
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 two µ-Slides VI 0.4 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 25: Serial Connection of Luer Slides for Flow Experiments (PDF)
Instructions µ-Slide VI 0.4 (PDF)
Instructions ibidi Pump System (PDF)

Material•••••••µ-Slide VI 0.4, ibiTreat (80606, 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)____________________________________________________________________________________
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Serial Connector (10830, ibidi)*
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.)* 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.
Then plug one plastic connector into
each end of the tubing. This results in aconnector tubing with two free male
Luer adapters at the ends to connect
channels between slides. Finally,sterilize the connector with ethanol orby autoclaving.

Figure 1: Example of an assembled SerialConnector.

1. PreparationsImportant Note: Equilibrate all material, such as the µ-Slide, 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.
The setup is equal to the standard experiment with only one channel. The only difference is thatyou connect multiple channels serially with each other before you connect the whole assembly to
the Perfusion Set.

1.1. Cell Seeding
Perform all the following protocol steps under sterile conditions!
Seed your cells as usual in the channels of µ-Slide VI 0.4 and let the cells attach. If further statical
incubation is needed, fill the reservoirs with 60 µl cell-free medium. A detailed description ofseeding cells is provided in the µ-Slide VI 0.4 Instructions, Application Note 3, “Cell Culture in ibidi
Channel Slides” and Application Note 13, “Endothelial Cells Under Perfusion”.
After cell attachment, the slide is ready for the serial connection procedure.

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). While running theprogram, gently tap on the tubes and the adapters with your fingers to remove the air bubbles. 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 FluidicUnit! 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 Channels of the µ-Slide VI 0.4
After cell seeding and attachment, follow these steps to connect the channels serially. Performthese steps as fast as possible to reduce cellular stress caused by temperature fluctuations 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-Slide 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.

2.1. Connecting the Serial Connectors to the µ-Slide VI 0.4•Put the µ-Slide Rack, the syringe, the equilibrated
medium, and the equilibrated Serial Connector into
the laminar flow hood. Place the µ-Slide 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 the male Luer adapter of one of the Serial
Connectors into one of the female Luer ports of thefirst channel of the µ-Slide (Fig. 2C). Twist theadapter while tightly pressing it into the Luer port.
Wipe off any overspilled medium using a paper
towel. The first serial connector is now connected tothe slide.

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Repeat this procedure with the other four Serial
Connectors and the four following channels,
alternating between front and back Luer ports (Fig.
2D–E) until all five serial connectors are connectedto the µ-Slide (Fig. 2F).

2.2. Connecting the Channels of the µ-Slide VI 0.4•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 channel of the µ-Slide. Make sure no air
bubbles are trapped inside (Fig. 3B). Wipe away
any spillover with a paper towel.

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Very carefully, push the medium through the firstchannel of the µ-Slide 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 free female Luer port of the second
channel of the µ-Slide (Fig. 3D).

•Keep the syringe connected to the µ-Slide.
Continue to push the medium through the channels
using the syringe, until the second Serial
Connector is filled entirely, and you see a hump ofliquid at the Luer adapter (Fig. 3E).

•Plug it into the free female Luer port of the next
channel (Fig 3F).

•Repeat the last two steps until all Serial
Connectors are plugged into the respective LuerPorts. If excessive medium has been spilled, wipe
if off using a paper towel. The channels of the µSlide VI 0.4 are now serially connected (Fig. 3G).

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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
to the Perfusion Set.
•Stop the flow of the ibidi Pump using the PumpControl
software. Next, disconnect the Fluidic Unit (with themounted Perfusion Set, filled with medium) from theibidi Pump and place it in the laminar flow hood close
to the µ-Slide VI 0.4 with serially connected channels.
Pinch off the Perfusion 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 the µ-Slide.
Slowly remove the syringe (Fig. 4D).

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

•Disconnect the second male Luer adapter from themiddle connector by holding it upwards (Fig. 4F).

•Connect the second male Luer adapter to theremaining female Luer port on the µ-Slide (Fig. 4G).

•The µ-Slide VI 0.4 with serially connected channels is
now connected to the Fluidic Unit. Wipe off any
excessive medium with a paper towel and remove thewhite plastic clamp (Fig. 4H).

Important Note: We recommend checking the cells
under the microscope before starting the perfusion
experiment.
It is crucial that the cell layer is confluent, and the cells
are well adherent when they are exposed to shear
stress. If the cells become stressed by the connecting
procedure, give them a few hours to recover in theincubator before starting the flow.
•Put the whole assembly into the incubator andconnect the Fluidic Unit to the ibidi Pump System.

•Start the flow experiment.

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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 belower 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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