3450-048-K
Contents
1. Relative invasion Test sample RFU Negative Control RFU Data is usually plotted in a bar graph as such amounts shown are per reactor Evaluation of Angiogeneis Activation Using DIVAA Relative Invasion INK E a 0 E neg CTRL 100 ng FGF 37 5 ng FGF 12 5 ng VEGF Data provided by John Basile 19 E1 3 11v1 VIII Troubleshooting Troubleshooting Guide Solution BME does not gel in angio reactor Variability in Assay High back ground in negative control Use a more concentrated compound formulation do not dilute BME more than 10 Use new BME Mix BME and test com pound thoroughly by gently pipeting up and down Do not use angioreactors taini ket Air pockets in angioreactor ike ae a Invert angioreactors when gelling Implant up to 2 angio reactors in each preformed pocket in dorsal flanks subcutaneously open end first inside pocket BME has been over diluted BME integrity has been compromised by inappropriate shipping storage or contamination Inadequate mixing of BME and test compound Improper implantation Allow cell surface receptors to recover for 1 hour by incubating cell in culture media containing 10 FBS Use nude mice Insufficient washing of cells Wash cells again in 1X Wash after FITC Lectin Staining Buffer Insufficient receptor recovery after CellSperse treatment Use of C57BI 6 mice Implantation period is too Reduce and optimize long2. Buffer to 10 ml using 14 Carefully remove the bottom cap of the angioreactors with a sterile razor deionized water and label DIVAA FITC Lectin Dilution Buffer l 20 For each angioreactor dilute 1 ul DIVAA 200X FITC Lectin to 200 ul blade and using a sterile 200 ul pipette tip push BME vessel complex g i H M g APE PE P using DIVAA FITC Lectin Dilution Buffer and label DIVAA FITC out of angioreactor into the sterile microtube See Figure 7 for vasculari Lectin zation in DIVAA Reduced Growth Factor BME plus FGF 2 VEGF 21 Resuspend pellet in 200 ul of DIVAA FITC Lectin and incubate at 4 C overnight 22 Centrifuge at 250 x g and remove supernatant Wash pellet three times in DIVAA Wash Buffer as indicated in step 12 E1 3 11v1 E1 3 11v1 23 Suspend pellet in 100 ul of DIVAA Wash Buffer for fluorometric deter mination 24 Measure fluorescence in 96 well plates excitation 485 nm emission 510 nm some fluorometers may require adjustment of Gain for an optimal range of values please consult your equipment user manual Optional Protocol for Calcein AM Detection not included in the DIVAA kit 1 2a After maintenance period humanely euthanize mice Exposure to CO2 levels greater than 70 for 5 minutes should be adequate Harvest angioreactors Remove a 2 cm perimeter of skin surrounding angioreactors using dissection scissors Using a scalpel cut along open end of angior
3. By William Stetler Stevenson Figure 4 Location of Incision Photo Provided By William Stetler Stevenson 10 Implant angioreactors into the dorsal flank of a mouse with the open end 11 opposite the incision up to 2 angioreactors may be planted on each side for a total of 4 angioreactors per mouse See Figure 5 for im plantation procedure and closure of the incision Distribute angio reactors with like pairs in each mouse see Figure 6 for recommended distribution Maintain mice for 9 to 15 days this step requires optimization Longer maintenance periods result in more vascularization E1 3 11v1 Figure 5 Implanting angioreactors For each mouse make a 5 mm incission on the posterior dorsal flank left and right and carefully insert surgical scissors to make a subcutaneous pocket Using forceps wet filled angioreactor in sterile 1X PBS to lubricate and insert angioreactor open end first into pocket up to two angioreactors can be placed in each pocket for a maximum of 4 angioreactors per mouse Close incission with skin staple and tag mouse for identification Place mice under heat lamp for 15 minutes to aid in recovery Photo Provided By William Stetler Stevenson E1 3 11v1 A Positive Control C Condition 1 E Condition 3 B Negative Control D Condition 2 F Condition 4 Figure 7 Vascularization in DIVAA angioreactor New vessel formation is appearant in the DIVAA RGF BME inside the angioreactor prio
4. and after 20 minutes humanely euthanize mice Exposure to CO2 levels greater than 70 for 5 minutes should be adequate Harvest angioreactors Remove a 2 cm perimeter of skin surrounding angioreactors using dissection scissors Using a scalpel cut along open end of angioreactor to sever any vessels that may be growing into it Recover angioreactor using dissection forceps Carefully remove the bottom cap of the angioreactors with a razor blade and using a sterile 200 uL pipet tip push BME vessel complex out of angioreactor into the sterile microtube See Figure 7 for vascu larization in DIVAA RGF BME with angiogenic factors 9 E1 3 11v1 4 Rinse inside of angioreactors with 300 ul of CellSperse into microtube Dispose of empty angioreactors Cap tube and incubate for 1 hour at 37 C 5 Clear incubation mix by centrifugation 15 000 x g for 5 minutes at room temperature 6 Measure fluorescence of supernatant in 96 well plates excitation 485 nm emission 510 nm some fluorometers may require adjustment of Gain for an optimal range of values please consult your equipment user manual VII Data Interpretation Values for cell invasion will be expressed in Relative Fluorescent Units RFUs Calculate the mean for each condition and its corresponding standard deviation Differences in conditions may be evaluated using a paired student s t test For inter assay comparison it may be more practical to compare relative invasion
5. implantation period Adjust gain on fluoro metric plate reader within optimal range Gain is improperly set on fluorometric plate reader i E1 3 11v1 No or low signal in positive control Mix BME and test compound thoroughly by gently pipeting up and down i l Do not use angioreactors Air pockets in angioreactor oe containing air pockets Invert angioreactors when gelling Implant up to 2 angio reactors in each pre formed pocket in dorsal flanks subcutaneously open end first inside pocket Inadequate mixing of BME and test compound Improper implantation Allow cell surface receptors to recover for 1 hour by incubating cell in culture media containing 10 FBS Insufficient receptor recovery after CellSperse treatment Omitting or inadequate mixing of Heparin in FGF 2 Add Heparin to FGF 2 and mix well before adding to BME Implantation period was not sufficient to elicit angiogenic Extend and optimize response implantation period Adjust gain on fluorometric plate reader within optimal range Gain is improperly set on fluorometric plate reader IX References 1 Guedez L Rivera AM Salloum R Miller ML Diegmueller JJ Bungay PM Stetler Stevenson WG 2003 Quantitative assessment of angiogenic response by the Directed n Vivo Angiogenesis Assay American J Pathol 162 1431 1439 2 Seo D Li H Guedez L Wingfield PT Diaz T Salloum R Wei B Stetler Stevenson WG 2003 TIMP 2 Mediated i
6. 560 4973 e mail info trevigen com www trevigen com E1 3 11v1
7. CULTREX Instructions For Research Use Only Not For Use In Diagnostic Procedures Directed In Vivo Angiogenesis Assay DIVAA TM Catalog 3450 048 K Table of Contents Section Title Page I Background 1 Il Precautions and Limitations 1 lil Materials Supplied 1 IV Materials Required But Not Supplied 2 V Reagent Preparation 2 n z n n VI Assay Protocols Directed In Vivo Angiogenesis A Preparing for Implantation 3 B Implanting Angioreactors 4 Assay DIVAA C FITC Lectin Detection 7 D Calcein AM Detection 9 E FiTC Dextran Detection 9 VII Data Interpretation 10 VIII Troubleshooting 11 Catalog 3450 048 K IX References 12 X Appendices A Reagent Composition 13 B Related Products from Trevigen 14 48 Samples 2011 Trevigen Cultrex PathClear and CultreCoat are registered trademarks and DIVAA CellSperse and AngioRack are trademarks of Trevigen Inc Teflon is a registered trade mark of Dupont Corporation i E1 3 11v1 I Background Please read the entire nstructions for Use prior to performing tests Trevigen s Directed n Vivo Angiogenesis Assay DIVAA is the first in vivo system for the study of angiogenesis that provides quantitative and reproducible results The DIVAA system was developed for and qualified using nude mice Therefore optimization will be necessary for normal mouse strains During the course of the assay implant grade silicone cylinders closed at one end called angior
8. eactor to sever any vessels that may be growing into it Recover angioreactor using dissection forceps Carefully remove the bottom cap of the angioreactors with a razor blade and using a sterile 200 ul pipette tip push BME vessel complex out of angioreactor into the sterile microtube See Figure 6 for vasculari zation in DIVAA RGF BME plus angiogenic factors Rinse inside of angioreactors with 300 ul of CellSperse into microtube Dispose of empty angioreactors Cap tube and incubate at 37 C to digest BME and create a single cell suspension This may take 1 3 hours Dilute 25 ml DIVAA 10X Wash Buffer to 250 ml using deionized water and label DIVAA Wash Buffer Centrifuge digested BME at 250 x g for 5 minutes at room temperature to collect cell pellets and insoluble fractions and discard supernatant Resuspend pellet in 500 ul of DIVAA Wash Buffer to wash cells and centrifuge again Discard supernatant and repeat wash two more times Add 100 ul of 1 uM Calcein AM in DIVAA Wash Buffer and incubate at 37 C for 60 minutes Measure fluorescence in 96 well plates excitation 485 nm emission 510 nm some fluorometers may require adjustment of Gain for an optimal range of values please consult your equipment user manual Optional Protocol for Dextran FITC Detection not included in DIVAA kit 1 After maintenance period inject 100 ul of 25 mg ml Dextran FITC in DIVAA Wash Buffer via tail vein
9. eactors are filled with 20 ul of Trevigen s basement membrane extract BME premixed with or without angiogenesis modulating factors These angioreactors are then implanted subcutaneously in the dorsal flanks of nude mice If filled with angiogenic factors vascular endothelial cells migrate into and proliferate in the BME to form vessels in the angioreactor As early as nine days post implantation there are enough cells to determine an effective dose response to angiogenic factors The sleek design of the angioreactor provides a standardized platform for reproducible and quantifiable in vivo angiogenesis assays Compared to the plug assay the angioreactor prevents assay errors due to absorption of BME by the mouse In addition the angioreactor uses only a fraction of the materials conserving both BME and test compounds used and up to four angioreactors may be implanted in each mouse giving more data for analysis Trevigen s DIVAA has been used in evaluating the inhibition of angiogenesis by TIMP 2 to study angiogenesis in matrix metalloprotease MMP 2 deficient mice and enhancement of angiogenesis associated with adrenomedullin and CD97 Trevigen s DIVAA was designed for assessing angiogenesis activation by test compounds and sufficient angiogenic factors are provided for 8 FGF 2 controls and 8 positive controls Il Precautions and Limitations 1 For Research Use Only Not for use in diagnostic procedures 2 The p
10. etamine HCL anesthesia 20 mg ml Xylazine analgesic Calcein AM FITC Dextran Angiogenic modulating factors except FGF 2 Ot a aN Disposables 1 Black 96 well fluorescence assay plate 2 Serological pipettes 3 Microscope slides and coverslips 4 Micropipettor tips V Reagent Preparation 1 10X Wash Buffer Dilute 25 ml of 10X Wash Buffer in 225 ml of sterile deionized water 2 FGF 2 100 ng Add 1 ul of Heparin Solution to 10 ul of FGF 2 100 ng and gently pipette up and down to mix immediately before addition to BME E1 3 11v1 3 FGF 2 300 ng VEGF 100 ng Add 1 ul of Heparin Solution to 10 ul of FGF 2 300 ng VEGF 100 ng and gently pipette up and down to mix immediately before addition to BME 4 25X FITC Lectin Diluent Dilute 400 ul of 25X FITC Lectin Diluent in 9 6 ml of sterile deionized water 5 200X FITC Lectin Dilute 50 ul of 200X FITC Lectin in 10 ml of 1X FITC Lectin Diluent VI Assay Protocol Note The entire procedure must be conducted under sterile conditions using aseptic technique to prevent contamination and subsequent infec tion in nude mice The use of normal mice will require optimization A Preparing Angioreactors for Implantation 1 Thaw Growth Factor Reduced BME at 4 C on ice overnight prior to assay BME is to be kept on ice until gelling in step 6 2 Pre chill all pipette tips angioreactors AngioRack Catalog 3450 048 09 sold separately and angiogenesis modula
11. he AngioRack Add 20 ul of BME with or without modulating factors to each angioreactor using a pre chilled sterile gel loading tip see Figure 1 Be careful not to introduce bubbles into the angioreactor One tube will fill eight angioreactors see Figure 2 Once the eight angioreactors are filled immediately invert angrioreac tors and transfer to a sterile microtube and place at 37 C for 1 hour to promote gelling inverting angioreactors during gelling prevents the formation of a meniscus at the open end of the angioreactor Repeat for the remainder of the angioreactors Figure 2 AngioRack containing filled angioreactors Implanting Angioreactors T Anesthetize each mouse immediately before implantation Recommen ded one part anesthesia 100 mg ml Ketamine HCL not included to four parts analgesic 20 mg ml Xylazine not included injected subcutaneously In a laminar flow hood using forceps remove angioreactor from micro tube cap and save microtube for step 6 See Figure 3 for implant preparation Incision should be made on the dorsal lateral surface of a nude mouse approximately 1 cm above the hip socket see Figure 4 Start by pinching back the skin and making a small cut using dissecting scissors Then extend cut to 1 cm in length being careful not to puncture under lying tissues E1 3 11v1 Figure 3 Preparing for implantation Arrange sterile instrumentation and anesthetize mouse Photo Provided
12. hysical chemical and toxicological properties of the products contained within the Directed In Vivo Angiogenesis Assay may not yet have been fully investigated Therefore Trevigen recommends the use of gloves lab coats and eye protection while using any of these chemical reagents Trevigen assumes no liability for damage resulting from handling or contact with these products MSDS sheets are available lll Materials Supplied Catalog Description Quantity Storage 3450 048 01 Angioreactors 48 units 4 C 3450 048 02 BME ao Factor Reduced 6 x 200 ul 20 C PathClear 3450 048 03 10X Wash Buffer 25 ml 4 C 3450 048 04 FGF 2 100ng 10 ul 20 C 3450 048 05 CellSperse 15 ml 20 C 3450 048 06 200X FITC Lectin 250 yg 50 ul 4 C 3450 048 07 25X FITC Lectin Diluent 400 ul 4 C 3450 048 08 Heparin Solution 10 ul 2 mg ml 4 C 3450 048 B9 FGF 2 300 ng VEGF 100 ng 10ul 20 C E1 3 11v1 IV Materials Equipment Required But Not Supplied Equipment 1 Mouse Cages Facility 2 Laminar Flow Hood or Clean Room 3 Pipette helper 4 Micropipettor 5 CQOz2 incubator 6 Fluorescent plate reader or microscope equipped with fluorescein long pass filter 7 500 ml graduated cylinder 8 Fine point forceps 10 Fine point cartilage forceps 11 Dissection scissors 12 Surgical scissors 13 Skin stapler 14 Scalpel 15 AngioRack Catalog 3450 048 09 sold separately Reagents Nude Mice Deionized water DMEM 10 FBS 100 mg ml K
13. mization Assay Cultrex 96 well BME Cell Invasion Assay Cultrex Laminin I Cell Invasion Assay Cultrex Collagen I Cell Invasion Assay Cultrex Collagen IV Cell Invasion Assay Cultrex 96 Well Cell Migration Assay Cultrex 24 Well Cell Migration Assay Description Cultrex Mouse Laminin Cultrex 3 D Culture Matrix Laminin Cultrex Rat Collagen Cultrex Bovine Collagen Cultrex 3 D Culture Matrix Collagen Cultrex Mouse Collagen IV Cultrex Human Fibronectin PathClear Cultrex Bovine Fibronectin Cultrex Human Vitronectin PathClear Cultrex Bovine Vitronectin Cultrex Poly D Lysine Cultrex Poly L Lysine Cultrex 3 D Culture Matrix BME Cultrex BME with Phenol Red PathClear Size 48 samples 48 samples 96 tests 96 tests 24 inserts 96 samples 96 samples 96 samples 96 samples 96 samples 12 samples Size 1mg 5ml 100 mg 50 mg 100 mg 1mg 1mg 1mg 50 ug 50 ug 100 mi 100 mi 15 ml 5 ml Cultrex BME with Phenol Red Growth Factor Reduced PathClear Cultrex BME PathClear Cultrex BME Growth Factor Reduced PathClear Cultrex Cell Staining Kit CellSperse 14 5 ml 5 ml 5 ml 100 ml 15 ml E1 3 11v1 The product accompanying this document is intended for research use only and is not intended for diagnostic purposes or for use in humans Trevigen Inc 8405 Helgerman Ct Gaithersburg MD 20877 Tel 1 800 873 8443 301 216 2800 Fax 301
14. nhibition of angiogenesis An MMP independent mechanism Cell 114 171 180 3 Martinez A Vos M Guedez L Kaur G Chen Z Garayoa M Pio R Moody T Stetler Stevenson WG Kleinman HK Cuttitta F 2002 The effects of adrenomedullin overexpression in breast tumor cells J Natl Cancer Inst 94 1226 37 1a E1 3 11v1 Wang T Ward Y Tian L Lake R Guedez L Stetler Stevenson WG Kelly K 2005 CD97 an adhesion receptor on inflammatory cells stimulates angiogenesis through binding integrin counterreceptors on endothelial cells Blood 105 2836 44 Lee MS Moon EJ Lee SW Kim MS Kim KW Kim YJ 2001 Angiogenic activity of pyruvic acid in in vivo and in vitro angiogenesis models Cancer Res 61 3290 3 Basile JR Holmbeck K Bugge TH Gutkind JS 2007 MT1 MMP controls tumor induced angiogenesis through the release of semaphorin 4D J Biol Chem 282 6899 905 X Appendices A Reagent Composition 1 Angioreactor Cat 3450 048 01 The angioreactor is a one centimeter long cylinder that is sealed on one end and houses 20 ul total volume It is made of implant grade silicone and provided sterile Angiogenesis is directed into the cylinder at the open end in response to angiogenesis modulating factors Growth Factor Reduced Basement Membrane Extract BME Cat 3450 048 02 BME is an extract from Engelbreth Holm Swarm EHS tumor composed primarily of Laminin I Collagen IV and Entactin BME provides an angiogenesis permissive matri
15. r to excission A and after harvest from the angioreactor B Photo Provided By William Stetler Stevenson 15 Rinse inside of each angioreactor with 300 ul of CellSperse and transfer into a microtube Dispose of empty angioreactors Cap tube and incubate at 37 C to digest BME and create a single cell suspension This may take 1 3 hours 16 Dilute 25 mL DIVAA 10X Wash Buffer to 250 mL using deionized water and label DIVAA Wash Buffer Figure 6 Recommended distribution of angioreacters in mice 17 Centrifuge digested BME at 250 x g for 5 minutes at room temperature to collect cell pellets and insoluble fractions and discard supernatant C FITC Lectin Detection Resuspend pellet in 500 ul of DMEM 10 FBS to allow for cell surface receptor recovery and incubate at 37 C for one hour 12 After maintenance period humanely euthanize mice Exposure to CO2 18 Centrifuge cells at 250 x g for 10 minutes at room temperature to collect levels greater than 70 for 5 minutes should be adequate cell pellets Resuspend pellet in 500 ul of DIVAA Wash Buffer to wash a pene a 2 mae permeter of Skin Surrounding angloreactors S gt g cells and centrifuge again Discard supernatant and repeat wash two dissection scissors Using a scalpel cut along open end of angioreactor to sever any vessels that may be growing into it Recover angioreactor more times using dissection forceps 19 Dilute 400 ul DIVAA 25X FITC Lectin Dilution
16. ting factors at 4 C and keep BME on ice 3 Working on ice add angiogenic factors to one tube 200 ul of Growth Factor Reduced BME Each tube of BME is sufficient for 8 angioreactors Add 10 ul of FGF 2 100 ng Cat 3450 048 04 or 10 ul of FGF 2 300 ng VEGF 100 ng Cat 3450 048 B9 and 1 ul of Heparin Solution per 200 ul of BME to use for the positive control angioreactors Add 11 uL of sterile PBS or test solvent per 200 ul BME to use for the negative control angioreactors 4 Still working on ice add test angiogenesis modulating factors to the remaining microtubes of Growth Factor Reduced BME do not add more than 10 total volume over diluting BME may compromise polymeri zation Gently pipette up and down to mix test or control factors and BME be careful not to introduce bubbles into the BME Bubbles may be Figure 1 Fill chilled 4 C angioreactors using a chilled 4 C gel loading tip from the bottom up Start with excess reagent 25 uL to prevent the introduction of bubbles insert capillary tip completely add BME and slowly withdraw pipet tip from angioreactor and fill to the top Fill 8 angioreactors at a time and procede to next step to prevent premature gelling 3 E1 3 11v1 B eliminated by centrifuging 250 x g for 5 minutes at 4 C Prepare to fill angioreactors Angioreactors must be kept chilled on ice prior to filling whether inside microtubes or situated in an AngioRack Place angioreactors in t
17. x for vessel formation in response to angiogenic factors 10X Wash Buffer Cat 3450 048 03 Proprietary buffer formulation CellSperse Cat 3450 048 05 A neutral metalloprotease from Bacillus polymyxa that provides for BME digestion and gentle cell dissociation 200X FITC Lectin Cat 3450 048 06 Fluorescence labeled Griffonia Simplicifolia Lectin binds to alpha p galactosyl and N acetyl galactosaminyl groups on the surface of endothelial cells 25X FITC Lectin Diluent Cat 3450 048 07 Proprietary buffer formulation Heparin Solution Cat 3450 048 08 2 mg mL Heparin 13 E1 3 11v1 9 FGF 2 300 ng VEGF 100 ng Cat 3450 048 B9 300 ng FGF and 100 ng VEGF B Related products available from Trevigen Catalog 3450 048 IK 3471 096 K 3470 096 K 3455 024 K 3484 096 K 3455 096 K 3456 096 K 3457 096 K 3458 096 K 3465 096 K 3465 024 K Accessories Catalog 3400 010 01 3446 005 01 3440 100 01 3442 050 01 3447 020 01 3410 010 01 3420 001 01 3416 001 01 3421 001 01 3417 001 01 3439 100 01 3438 100 01 3445 048 01 3430 005 02 3431 005 02 3432 005 02 3433 005 02 3437 100 K 3450 048 05 Description 3450 048 SK Cultrex DIVAA Starter Cultrex DIVAA Inhibition Kit Cultrex In Vitro Angiogenesis Assay Endothelial Cell Invasion Kit Cultrex In Vitro Angiogenesis Assay Tube Formation Kit 24 Well BME Cell Invasion Assay CultreCoat 96 well BME Coated Cell Invasion Opti
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