Sample Data Sheet
Contents
1. Photo Provided By William Stetler Stevenson C A Positive Control C B Condition 1 E Condition 3 Negative Control D Condition 2 F Condition 4 Figure 6 Recommended distribution of angioreacters in mice FITC Lectin Detection 12 After maintenance period humanely euthanize mice Exposure to CO2 13 14 levels greater than 70 for 5 minutes should be adequate 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 sterile razor blade and using a sterile 200 ul pipette tip push BME vessel complex out of angioreactor into the sterile microtube See Figure 7 for vasculari zation in DIVAA Reduced Growth Factor BME plus FGF 2 VEGF Figure 7 Vascularization in DIVAA angioreactor New vessel formation is appearant in the DIVAA RGF BME inside the angioreactor prior to excission A and after harvest from the angioreactor B Photo Provided By William Stetler Stevenson 15 16 17 18 19 20 21 22 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 Dilute2. 8 angioreactors at a time and procede to next step to prevent premature gelling 3 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 the 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 ab
3. Invasion Assay 96 samples 3456 096 K Cultrex Laminin I Cell Invasion Assay 96 samples 3457 096 K Cultrex Collagen Cell Invasion Assay 3458 096 K Cultrex Collagen IV Cell Invasion Assay 96 samples 3465 096 K Cultrex 96 Well Cell Migration Assay 96 samples 3465 024 K Cultrex 24 Well Cell Migration Assay 12 samples Accessories Catalog Description Size 3400 010 01 Cultrex Mouse Laminin 1mg 3446 005 01 Cultrex 3 D Culture Matrix Laminin 5 ml 3440 100 01 Cultrex Rat Collagen 100 mg 3442 050 01 Cultrex Bovine Collagen 50 mg 3447 020 01 Cultrex 3 D Culture Matrix Collagen 100 mg 3410 010 01 Cultrex Mouse Collagen IV 1mg 3420 001 01 Cultrex Human Fibronectin PathClear 1mg 3416 001 01 Cultrex Bovine Fibronectin 1mg 3421 001 01 Cultrex Human Vitronectin PathClear 50 ug 3417 001 01 Cultrex Bovine Vitronectin 50 ug 3439 100 01 Cultrex Poly D Lysine 100 ml 3438 100 01 Cultrex Poly L Lysine 100 mi 3445 048 01 Cultrex 3 D Culture Matrix BME 15 ml 3430 005 02 Cultrex BME with Phenol Red PathClear 5 mi Cultrex BME with Phenol Red Growth Factor ele Reduced PathClear ami 3432 005 02 Cultrex BME PathClear 5ml 3433 005 02 Cultrex BME Growth Factor Reduced PathClear 5 mi 3437 100 K Cultrex Cell Staining Kit 100 ml 3450 048 05 CellSperse 15 ml The product accompanying this document is intended for research use only and is not intended for diagnostic purposes or for use in human
4. 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 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 IN E a 0 E neg CTRL 100 ng FGF 37 5 ng FGF 12 5 ng VEGF Data provided by John Basile 10 VIII Troubleshooting Troubleshooting Guide Solution BME does not gel in angio reactor Variability in Assay BME has been over diluted BME integrity has been compromised by inappropriate shipping storage or contamination Inadequate mixing of BME and test compound Air pockets in angioreactor Improper implantation Insufficient receptor recovery after CellSperse treatment 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 containing air pockets Invert angioreactors when gelling Implant up to 2 angio reactors in each preformed pocket in dorsal flanks subcutaneously open end first inside pocket Allow cell surface receptors to recover for
5. 1 hour by incubating cell in culture media No or low signal in positive control Inadequate mixing of BME and test compound Mix BME and test compound thoroughly by gently pipeting up and down l Do not use angioreactors Air pockets in angioreactor ope containing air pockets Invert angioreactors when gelling Improper implantation Insufficient receptor recovery after CellSperse treatment Omitting or inadequate mixing of Heparin in FGF 2 Implantation period was not sufficient to elicit angiogenic response Implant up to 2 angio reactors in each pre formed pocket in dorsal flanks subcutaneously open end first inside pocket Allow cell surface receptors to recover for 1 hour by incubating cell in culture media containing 10 FBS Add Heparin to FGF 2 and mix well before adding to BME Extend and optimize implantation period containing 10 FBS Use nude mice Insufficient washing of cells Wash cells again in 1X Wash after FITC Lectin Staining Buffer Adjust gain on fluorometric plate reader within optimal range Gain is improperly set on Use of C57BI 6 mice fluorometric plate reader IX References High back Implantation period is too Reduce and optimize ground in long implantation period l negative control 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 A
6. 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 DMEM 10 FBS to allow for cell surface receptor recovery and incubate at 37 C for one hour Centrifuge cells at 250 x g for 10 minutes at room temperature to collect cell pellets Resuspend pellet in 500 ul of DIVAA Wash Buffer to wash cells and centrifuge again Discard supernatant and repeat wash two more times Dilute 400 ul DIVAA 25X FITC Lectin Dilution Buffer to 10 ml using deionized water and label DIVAA FITC Lectin Dilution Buffer For each angioreactor dilute 1 ul DIVAA 200X FITC Lectin to 200 ul using DIVAA FITC Lectin Dilution Buffer and label DIVAA FITC Lectin Resuspend pellet in 200 ul of DIVAA FITC Lectin and incubate at 4 C overnight Centrifuge at 250 x g and remove supernatant Wash pellet three times in DIVAA Wash Buffer as indicated in step 12 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 D
7. CULTREX Instructions For Research Use Only Not For Use In Diagnostic Procedures Directed n Vivo Angiogenesis Assay DIVAA Catalog 3450 048 K Table of Contents Section Title I Background Il Precautions and Limitations lll Materials Supplied DI IAA IV Materials Required But Not Supplied V Reagent Preparation Vi Assay Protocols Directed n Vivo Angiogenesis A Preparing for Implantation B Implanting Angioreactors Assay DIVAA C FITC Lectin Detection D Calcein AM Detection E FiTC Dextran Detection Vil Data Interpretation VIII Troubleshooting Catalog 3450 048 K IX References X Appendices A Reagent Composition B Related Products from Trevigen 48 Samples AMSBIO www amsbio com info amsbio com ae UK amp Rest of the World North America Germany ANS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 1 617 945 8218 U ClO ON BR WwW Nh NH a a a 2 11 12 13 14 Switzerland Centro Nord Sud 2E CH 6934 Bioggio Lugano T 41 0 91 604 55 22 F 41 0 91 605 17 85 I Background Please read the entire nstructions for Use prior to performing tests Trevigen s Directed n Vivo Angiogenesis Assay DIVAA i
8. IVAA kit 1 2 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 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 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 fluoromet
9. ers 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 and after 20 minutes humanely euthanize mice Exposure to COz 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 amsbio 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
10. ove 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 Figure 3 Preparing for implantation Arrange sterile instrumentation and anesthetize mouse Figure 4 Location of Incision Photo Provided By William Stetler Stevenson 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 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
11. quipped 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 Ketamine HCL anesthesia 20 mg ml Xylazine analgesic Calcein AM FITC Dextran Angiogenic modulating factors except FGF 2 O N wl ce gt 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 2 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 no
12. rmal 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 modulating 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
13. s AMSBIO www amsbio com info amsbio com SS UK amp Rest of the World g North America Germany AES 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 1 617 945 8218 Switzerland Centro Nord Sud 2E CH 6934 Bioggio Lugano T 41 0 91 604 55 22 F 41 0 91 605 17 85
14. s 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 angioreactors 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 7 to study angiogenesis in matrix metalloprotease MMP 2 deficient mice and enhancement of angiogenesis a
15. sis 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 matrix 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 amsbio 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 Description Size 3450 048 SK Cultrex DIVAA Starter 48 samples 3450 048 IK_ Cultrex DIVAA Inhibition Kit 48 samples 3471 096 K Cultrex In Vitro Angiogenesis Assay Endothelial Bea ee Cell Invasion Kit 3470 096 K Cultrex In Vitro Angiogenesis Assay Tube Seca Formation Kit 3455 024 K 24 Well BME Cell Invasion Assay 24 inserts 3484 096 K ahaa 96 well BME Coated Cell Invasion de sanipiss ptimization Assay 3455 096 K Cultrex 96 well BME Cell
16. ssay American J Pathol 162 1431 1439 Adjust gain on fluoro metric 2 Seo D Li H Guedez L Wingfield PT Diaz T Salloum R Wei B Stetler plate reader within optimal Stevenson WG 2003 TIMP 2 Mediated inhibition of angiogenesis An MMP range 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 Nat Cancer Inst 94 1226 37 11 12 Gain is improperly set on fluorometric plate reader 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 angiogene
17. ssociated 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 physical 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 ea 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 ug 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 amsbio 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 CQz2 incubator 6 Fluorescent plate reader or microscope e
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