Prtcl Preparatin Prtcl fr the Nn-Targeted Vev MicrMarker Cntrast Agent
System Cmpatibility: This guide cntains instructins and suggestins fr wrk n the Vev2100, VevLAZR, Vev 3100 systems and transducers frm the MS, MZ and MX series. Intrductin The Vev MicrMarker cntrast agents are manufactured by Bracc Research SpA and used t enhance tissue and vascular imaging exclusively n the VisualSnics Vev high-frequency ultrasund systems: Vev MicrMarker agents are lyphilized micrbubbles with a lipid-based uter shell that cntain plyethylene glycl, phsphlipids and fatty acids; Vev MicrMarker agents are stred in a glass vial cntaining a gas head-space cnsisting f nitrgen and perflurbutane; The micrbubbles will becme gas-filled agents when recnstituted with saline; Vev MicrMarker vials can be stred at 4 C (39.2 F) and have a shelf life f 12 mnths; Vev MicrMarker agents that have been pened and recnstituted are stable within the vials fr 4-6 hurs; Vev MicrMarker Nn-Targeted Cntrast Agent has a clearance time f apprximately 15-20 minutes in mice. Objective The bjective f this prtcl is t utline instructins fr the preparatin f Vev MicrMarker Nn- Targeted cntrast agent t be administered via blus injectin r via an infusin pump. ***Nte***: This is a generic prtcl describing the preparatin f the cntrast agent and blus injectin int the subject. Refer t applicatin-specific prtcls fr details abut imaging and data analysis using the Vev systems. This prtcl is intended fr preclinical imaging applicatins nly. Page 2 f 6
Materials needed fr the preparatin f Nn-Targeted Cntrast Agent 3x1 ml syringe 2x21G 5/8 needle 2x27G 1/2 needle Sterile saline vial Vev MicrMarker Nn-Targeted cntrast agent 1 vial Additinal materials nt prvided in the kit that may be required include: Additinal 1 ml syringes Additinal 21G 5/8 needles Additinal 2x27G 1/2 needles Venus Cannulatin device Sterile Saline Eppendrf tubes Pipettes and tips Infusin Pump the fllwing vendrs have varius mdels f suitable pumps available - www.syringepump.cm - www.harvardapparatus.cm Preparatin A Prepare Vev MicrMarker Cntrast Agent fr Muse Imaging 1. Attach the 21G 5/8 needle t the 1 ml syringe; 2. Recnstitute the cntrast agent by injecting 0.7 ml f saline int the Vev MicrMarker vial. Remve the syringe and leave the needle in the vial t vent fr a few secnds, and then remve the needle cmpletely. 3. Gently agitate the vial by hand fr ne minute. 4. Let it rest fr 10 minutes at rm temperature. B Preparatin fr Blus Injectin f Cntrast Agent 1. Gently agitate the vial in a tp-t-bttm manner befre cllecting a sample. Using a new 1 ml syringe and 21G 5/8 needle, draw up apprximately 120 µl f prepared Vev MicrMarker cntrast agent frm the vial t cmpensate fr the dead space in the needle hub. Ensure that all f the 120 µl f cntrast agent is withdrawn int the syringe, leaving nthing in the needle hub. 2. Replace the 21G 5/8 needle with the 27G 1/2 needle t prepare fr blus injectin. Remve the air that was drawn up and adjust the plunger t the vlume mark f 50 µl. This is the blus amunt t be injected int the cannulatin set-up (the cntrast syringe ). Nte: The blus shuld nly be prepared when all ther preparatin steps have been cmpleted and shuld be injected int the muse immediately upn remval f the cntrast agent frm the vial t prevent micrbubble destabilizatin. Page 3 f 6
C Vev MicrMarker Cncentratin and Recmmended Dilutin Methds 1. The recnstituted Vev MicrMarker vial has a cncentratin f 2x10 9 micrbubbles/ml. 2. A 50 µl blus will give a final cunt f 1.0 x10 8 micrbubbles, which is ptimal fr mst tumr imaging and less vascularized areas like the hind limb. 3. Fr lwer cncentratins the cntrast agent can be further diluted with saline in rder t perfrm a dse respnse curve r t determine the ptimal number f micrbubbles fr the applicatin. Fr example, 1.0 x10 7 micrbubbles/50 µl is recmmended fr well vascularized tissues such as the liver. This can be achieved by perfrming a 1:10 dilutin f the riginal stck cncentratin. Fr this dilutin, it is recmmended t mix 15 µl f cntrast t 135 µl f saline. Ensure dilutins are gently mixed immediately prir t the injectin. ***Nte***: Dilutin reduces the stability f the cntrast agents. Therefre, any diluted cntrast shuld be discarded if nt used within a few minutes frm time f the dilutin prcedure; further any unused stck slutin remved frm the vial shuld als be discarded. Recmmended blus injectin cncentratins fr specific target anatmies in mice: Target Anatmy Cncentratin Tumrs 1 x 10 8 bubbles/50 µl blus Retina 1 x 10 8 bubbles/50 µl blus Cardivascular 1 x 10 7 bubbles/50 µl blus Kidney 1-5 x 10 7 bubbles/50 µl blus Liver 1 x 10 7 bubbles/50 µl blus Pancreas 1 x 10 7 bubbles/50 µl blus Hind Limb (muscle) 1 x 10 8 bubbles/50 µl blus Heart 0.1-1 x 10 7 bubbles/50 µl blus Page 4 f 6
D Preparatin f Syringe Pump 1. Prgram the syringe pump fr 20-100 µl per minute delivery (see Table 1 fr recmmended rates fr varius applicatins). 2. Ensure the prper syringe diameter is prgrammed int the syringe pump. 3. Cnnect the stck injectin syringe int the cannulatin set-up. 4. Manually and carefully prime the cannulatin tubing with Vev MicrMarker cntrast agents. 5. Place the stck injectin syringe int the syringe pump. 6. Start the infusin pump t begin experimentatin. Recmmended infusin rates fr varius target anatmies in mice: Target Anatmy Infusin Rate Tumrs Retina Cardivascular Kidney Liver Pancreas Hind Limb (muscle) 5 µl/min 20 µl/min 20 µl/min Nte: Ultrasund cntrast agents tend t separate thus micrbubbles may flat and cllect at the tp f the syringe. Gentle rlling agitatin f the whle syringe pump/stck syringe set-up may assist in keeping the micrbubbles suspended in the slutin during the infusin. This can be dne by hand r an apprpriate mechanical device. Page 5 f 6
FUJIFILM VisualSnics, Inc. T.1.416.484.5000 Tll Free (Nrth America) 1.866.416.4636 Tll Free (Eurpe) +800.0751.2020 E. inf@visualsnics.cm www.visualsnics.cm VisualSnics, Vev, MicrMarker, RMV, EKV, MicrScan, Insight Thrugh In Viv Imaging are trademarks and registered trademarks f FUJIFILM SnSite, Inc. in varius jurisdictins. All ther trademarks are the prperty f their respective wners. Page 6 f 6