USER MANUAL
Microbubble Reagent for Contrast Ultrasound Applications

VesselVueTM P-formulation
Part number:P-4007003

Vial Size:

3 mL

Volume:1 mL

Storage:4°C

Version: 2.2

For Research Use Only and not for therapeutic or diagnostic use

VesselVue
Microbubble Regent for Contrast Ultrasound Applications
Caution: For Research Use. This product is intended for animal research only and not for use inhumans.
Part Number: P-4007003
Use with Revvity’s Bead RuptorTM VesselVueTM Homogenizer, Part Number: 25-030

Product Description
VesselVueTM formulations are microbubble contrast reagents for contrast enhanced
ultrasound (CEUS) imaging and preclinical therapy applications. VesselVue microbubbles canenable the visualization and quantification of tumor or organ vascularity in CEUS imaging,revealing dynamic changes in blood vessels for disease characterization and therapeutic
response studies. In addition to diagnostic research applications, some labs have also used
VesselVue microbubbles as either a catalyst or a delivery modality to interrogate several
different ultrasound-enabled therapeutic applications in preclinical studies.

P-formulation (Polydisperse):•••Has a wider microbubble size distribution, and a smaller mean diameter.
This formulation is an excellent all-around vascular imaging contrast agent for
general CEUS imaging applications.
This formulation can provide improved imaging signal sensitivity for specific CEUS
applications. The larger bubble size also increases the circulation time for bolus
injections, extending the imaging window.

This guide is for preparing the VesselVue P-formulation agent for use in traditional CEUS
imaging, or in conjunction with Acoustic Angiography, a CEUS imaging mode exclusive to theVega® Automated Ultrasound Imaging System, to help users achieve high-sensitivity in vivo
ultrasound imaging of microvasculature.

Each 3 mL vial contains 1 mL of P-formulation contrast agent, sufficient for imaging approx. 520 mice (weight ~20 grams each) when diluting 1:1 with saline via either a bolus or continuous
infusion I.V. injection.

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Storage and Handling
VesselVue vials are shipped on cold gel packs. Immediately after arrival, store vials at 4° C
until ready to use. Never freeze.
When stored as directed, VesselVue P-formulation remains viable for a minimum of 6 months
from the date of shipping.
VesselVue is best when used within 24 hours after septum has been pierced. Vials can be used
for multiple injections within a single imaging study. Pierced vials should not be stored. Discard
unused portion.

Administration Methods and Preparation
Decide in advance which administration method you will use.
Here we briefly describe two methods. Most studies in literature use tail vein infusion for
microbubble contrast agents in rodents. Some studies have also reported using I.V. injection
via other peripheral veins.
I.

Bolus

The half-life, or circulation, time for P-formulation microbubbles is 2-5 minutes whenusing bolus injections [Sirsi 2010]. The VesselVue contrast solution is diluted1:1 withsaline for a final volume of 100 μL per mouse. Maximum volume is determined byyour lab’s specific IACUC protocol.
•II.

A larger dose can be injected initially to extend the imaging window. However,
too many microbubbles in circulation may result in decreased image quality, as
the layer of microbubbles will “shadow” regions of tissue below the skin’ssurface.

Continuous Infusion

For continuous infusion, a “tail vein catheter” consisting of a syringe connected to athin gauge of medical grade plastic tubing is coupled to the tail vein of a mouse orrat. The syringe contains VesselVue microbubbles diluted 1:1 with saline.
•For guidance on creating tail vein catheters, please contact our Global
Technical Support Team: LS.ReagentsTechSupport@revvity.com

More detail is provided on performing each of the two methods in the following pages of thisprotocol.
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1. Preparing Mice for Imaging
The fur on and around the areas of the animal that are to be imaged should bedepilated prior to microbubble injection. Nude mice, or immunocompetenthairless SKH1-E mice, do not require depilation; however conventional strains ofhaired mice, like BALB/c or C57BL/6, require depilation.

2. Preparing VesselVue Microbubbles
VesselVue microbubbles are shipped unactivated, meaning you must activate them
immediately prior to use with the Bead Ruptor
VesselVue Homogenizer.

Above: P-formulation agent
shown before and after activation
Below: The Bead Ruptor

VesselVue Homogenizer, 25-030

Speed: 5
Time: 90

Activation should only be performed once on an
unopened vial just prior to use. Allow the vial towarm to room temperature before placing inhomogenizer.
To activate the microbubbles, insert anunopened vial (unpierced septum) into the BeadRuptor VesselVue Homogenizer vial holder.
Secure the vial using the retainer screw andclose the cover.
Adjust the Speed setting to 5
Adjust the Time setting to 90 seconds
Press the Start button.
When mixing cycle is complete, loosen theretaining screw that secures the vial in theholder and remove the vial from thehomogenizer.

Let the vial sit for 5 minutes to allow the microbubble concentration to stabilize, then proceed

to administration protocol below.
For more information, refer to the Bead Ruptor VesselVue Vial Homogenizer User Manual.

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3. Administration and Imaging Protocols
P-FORMULATION AGENT MUST BE ACTIVATED BEFORE PROCEEDING TO EITHERADMINISTRATION METHOD BELOW.

A 1:1 microbubble/saline ratio is typically used for the Acoustic Angiography
imaging mode on the Vega ultrasound system. Other dilutions have been
published and can be tailored to your specific instrumentation or research
application.
Note that when withdrawing stock microbubbles during the dilution process,care must be taken to avoid damaging the microbubbles by introducing suddenchanges in pressure (vacuum or compression).
Microbubbles should be diluted with sterile saline immediately prior to use.

I. Preparation for a Bolus Administration
A bolus of microbubbles can be injected through the tail vein.
Using a 1:1 microbubble/saline solution, the recommended total volume is:••

100 μL per mouse (20 g)
150 μL per rat (~150 g)

1. Allow the vial to warm to room
temperature before using theVesselVue mixer, and let the vial sit
for 5 minutes after mixing to allow
the microbubble concentration tostabilize.
2. Add appropriate amount* of sterile
saline to a 1- 5 mL syringe using a
20g needle or similar (*Maximum
volume is determined by your lab’s
specific IACUC protocol). Wait until
shortly before imaging to continue tonext steps.
3. While the VesselVue vial is upright,
insert a venting needle into theseptum. The vent needle is criticalto equalize the internal airpressure with the atmosphere. (Fig1A)

VesselVue P-formulation User Guide V2.2

Figure 1. (A) Insert two needles into the rubber septa. (B)Invert the vial and draw the contrast agent into the syringe
while allowing the vial to vent to prevent vacuum formation.
Failure to prevent vacuum in the vial will destroy
microbubbles.

44. Insert the extraction needle connected to the saline syringe into the septum next tothe venting needle. (Fig. 1A)
5. Invert the vial and ensure that the tip of the venting needle reaches above themicrobubble/gas interface. (Fig 1B)
6. Draw the appropriate volume of microbubbles into the syringe, ensuring that the tipof the injection needle remains below the microbubble/gas interface. (Fig 1B)
7. Mix solution by gently rolling the syringe between the palms of your hands. DO NOT
mix by pumping the syringe.

II.

Preparation for a Continuous Infusion Administration
Suggested Materials:•••••••1:1 diluted VesselVue P-formulation microbubble contrast agent (MinimumCalculated Volume)
VesselVue Microbubble Mixer
Catheter set, including thin gauge medical grade plastic tubing
Sterile saline for priming the catheter to prevent air from being introduced
into the vein, and to confirm successful needle placement in vein
2 syringes: one for normal saline and one for the diluted VesselVue contrast
reagent
Medical tape or other biocompatible adhesive to secure the needle
placement at the injection site
Syringe pump for administration of diluted VesselVue

1. Allow the vial to warm to room temperature before using the VesselVue mixer, andlet the vial sit for 5 minutes after mixing to allow the microbubble concentration tostabilize.
2. To set up catheter infusion line, prepare one syringe containing saline and one
syringe containing 1:1 microbubble dilution mounted on a syringe pump with correct
settings for speed. Place pump close to instrument to keep length of the catheter
short. Keep the overall length of the catheter tube as short as possible to reducethe volume of saline in the catheter.
3. Prime the tubing with sterile saline by manually injecting enough saline to fill thecatheter. Also, take this opportunity to estimate the dead space in the catheter set(i.e. the volume in the needle and tubing combined).
4. Connect the syringe containing the calculated volume to the pre-filled tubing
coupled to the animal’s tail vein via a needle. Secure the tubing in place and start
the pump.
5. Wait approximately 30 seconds before starting image acquisition in order for the
microbubbles to move through the tubing and achieve a steady state level ofmicrobubbles in the animal’s vasculature (perfusion time).
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Minimum Calculated Volume
The calculated volume of microbubbles and saline needed for infusion per mouse isdetermined by three variables. These are:
1.
2.
3.

Dead-space in syringe and catheter (catheter volume)
Estimated scan time
Imaging subject (mouse or rat)

1. Dead-space in syringe and catheter (catheter volume)a. The catheter volume can be determined using saline and will depend on the length oftubing used.
Note: be mindful of the length of the tubing, as a non-negligible amount of microbubble

solution will occupy the tubing and decrease the microbubble solution volume available
for imaging in longer imaging studies. Longer tubing also increases the pressure in thetube which could damage the bubbles.
2. The estimated scan time is determined by the size of the area to be scanned (larger area =longer scan time) and the preset mode selected in the software. The modes have acorresponding length of time needed to scan for that selected preset.
a. The Vega Ultrasound Imaging System’s software, SonoEQ, includes 2 parameter“presets” for Acoustic Angiography depending on your imaging goals:
Density: captures signal from both sub-resolution and resolvable vessels.
Morphology: eliminates sub-resolution signal to highlight resolvable vessels.

An estimate of the scan time required for each preset is provided in SonoEQ (Please
see the SonoEQ user manual).

3. Whether the imaging subject is a mouse or a rat. Infusion rates are:••

15 μL/min per mouse (20 g)
40 μL/min per rat (~150 g)

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Example of Minimum Calculated Volume:
Using the infusion rate for a mouse, the following example applies to a Morphology preset
scan that takes 4 min and requires 100 μL to fill the catheter.
(15 μL/min mouse injection rate) * (4.0 min scan time)+ (15 μL/min mouse injection rate) * (0.5 min perfusion time)+ (100 μL pre-fill catheter volume)= ~170 μL total volume injected per mouse
Note: 170 μL total volume is 85 μL saline and 85 μL of microbubbles, assuming adilution of 1:1.
For further information on in vivo imaging and related products, please contact your local
Revvity representative or visit: https://www.revvity.com
For Technical Support, please contact: LS.ReagentsTechSupport@revvity.com

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