OrisTM 3D Embedded Invasion Assay
Contents
1. Add the Collagen Matrix solution to fill the detection zone column and incubate for 1 hour to allow for collagen polymerization Add 100 uL of culture medium to each well and place the plate back in the incubator Monitor periodically to assess cell movement Upon completion of the assay use a microscope to examine each well to determine the optimal cell seeding concentration for your specific cell type APPENDIX Il Fluorescent Labeling Live Cell Options It is important to label cells using a fluorescent reagent that uniformly stains cells Please consult the manufacturer of your fluorescent stain for specific considerations SrA lt 2 NOTE Use caution when adding removing solutions so that the Collagen Matrix is not dislodged from the bottom sides of the well The following is an example Fluorescent Staining Protocol to label live cells with Calcein AM a O xA Oe NA c d e f To stain one fully seeded 96 well plate combine 5 uL of Calcein AM 1 mg mL in anhydrous DMSO with 10 mL of phenol red free and serum free medium or 1x PBS containing both Ca and Mg Protect diluted Calcein AM solution from light until ready to use in step d Carefully remove the culture medium from wells with a pipette Wash wells with 100 uL of PBS containing both Ca and Mg Add 100 uL of diluted Calcein AM solution to each well Incubate plate at 37 C for 30 60 minutes Attach mask and read promptly with microp2. Fluorescence Microplate Reader optional Cell Culture Labeling Medium phenol red free serum free Cell Labeling Fluorescent Agent e g Calcein AM required if performing staining Oris is a trademark of Platypus Technologies LLC Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 PRECAUTIONS AND RECOMMENDATIONS For Research Use Only Not for use in diagnostic procedures Handling and Use of the Oris Collagen I Rat tail e Be sure to keep Oris Collagen Rat tail on ice while being used e A suggested concentration for the Oris Collagen I Matrix is 3 mg mL e f you prefer to use a different concentration of the Collagen Matrix solution for your particular cell line be sure to adjust the volumes of the other associated reagents in proportion to the adjustments made to the volume of the collagen Recommendations for Preparation of Reference Wells Establishing a time zero t 0 reference can be accomplished by any of several methods real time analysis treating reference wells with a migration inhibitor or utilizing the stopper as a physical barrier to prevent cell movement into the detection zone e Real time analysis using microplate reader treat the cells with a live fluorescent stain at the start of the assay Attach the Oris Detection Mask to the bottom of the plate see Section IV
3. Step 5 Quantify fluorescence using a microplate reader This data is the pre invasion reference value e Real time analysis using microscopy with imaging capabilities images collected at the start of the experiment can be used as the t 0 reference to be analyzed compared with subsequent images Inhibitor treatment treat control wells with a concentration of drug that fully inhibits cellular movement Choice of drug and concentration will be cell line dependent To ensure the correct drug concentration you may need to test a range of drug concentrations in a separate experiment Oris Cell Seeding Stoppers as barrier NOTE Stoppers come in strips of n 4 Set up the assay as normal following the protocol in Section IV For the t 0 reference wells skip steps 12 14 Continue with the rest of the experiment as normal The stoppers can remain in place as a t 0 reference until the assay is complete Upon completion the stoppers may then be removed to allow for endpoint fixation and labeling Recommendations for 10X PBS Buffer e When 10X PBS is refrigerated sedimentation may occur due to the high salt concentration If sediment forms warm the PBS in a water bath 37 C to completely dissolve any sediment prior to use Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 ee SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 3 IV Oris 3D EMBEDDED INVASION ASSAY
4. P L AT YP UX Bringing Science to the Surface Oris 3D Embedded Invasion Assay Product No EIAST 3D Assay for Investigating Embedded Cell Movement through Collagen PROTOCOL amp INSTRUCTIONS MATERIALS PROVIDED MATERIALS REQUIRED PRECAUTIONS AND RECOMMENDATIONS Oris 3D EMBEDDED INVASION ASSAY PROTOCOL DATA ACQUISITION Oris PLATE DIMENSIONS ORDERING INFORMATION VIII TERMS amp CONDITIONS APPENDIX I Determining Optimal Cell Seeding Concentration APPENDIX II Fluorescent Labeling Live Cell Options APPENDIX IIl Fluorescent Labeling Fixed Cell Options Platypus Technologies LLC 5520 Nobel Drive Suite 100 Madison WI 53711 Toll Free 866 3296 4455 Phone 608 237 1270 Fax 608 237 1271 www platypustech com SP0232 01 Oris 3D Embedded Invasion Assay Important Read Instructions Before Performing any Oris Assay I MATERIALS PROVIDED Product No EIAST Oris compatible 96 well Collagen Oris Cell Seeding Stoppers Refrigerate 4 C Oris Detection Mask Oris Stopper Tool Oris Collagen Rat tail Oris Collagen Rat tail must be stored at 4 C for use within 6 months of receipt Do not freeze ll MATERIALS REQUIRED e Cells 7 5 Sodium Bicarbonate Cell Culture Growth Medium Sterile PBS e Serum Free Cell Culture Medium Sterile Pipette Tips Pipette or Multi Channel Pipette Trypsin or Cell Scraper Inverted Microscope optional
5. PROTOCOL The following steps should be performed in a biological hood using aseptic technique to prevent contamination 1 If desired cells can be starved by incubating for 18 24 hours in serum free medium prior to assay Alternatively 0 5 fetal bovine serum may be used if needed Remove the Oris compatible 96 well Collagen Coated Plate from refrigeration and allow to equilibrate to room temperature for one hour 3 Populate the 96 well plate with Oris Cell Seeding Stoppers e Vertically position the tip ends of two 4 stopper strips into one full column of 8 wells at a time Figure 1A e Gently press down on the strip backbone to partially insert the stopper halfway into the well Figure 1B e When both stopper strips have been partially inserted in one column ensure that the position of the stoppers is vertical with respect to the well wall making any necessary adjustments Figure 1C e Using the Oris Stopper Tool firmly press down on the strip backbone to fully insert the stoppers into each well Figure 1D and 1E Repeat for all remaining stoppers t lt NOTE It is extremely important to ensure that the stopper are 2 inserted perpendicular to the well bottom and are fully engaged with the well bottom If you require the stoppers to Figure 1 Stopper Insertion Process A Placement of Stoppers be more consistently located the pre populated Oris 3D into Wells B Close up of Stoppers Partially Inserted in
6. Y KIND FOR ANY INDIRECT SPECIAL INCIDENTAL CONSEQUENTIAL OR EXEMPLARY DAMAGES INCLUDING BUT NOT LIMITED TO LOST PROFITS EVEN IF PLATYPUS HAD NOTICE OF THE POSSIBILITY OF SUCH DAMAGES Without limiting the effect of the preceding sentence PLATYPUS s maximum liability if any shall not exceed the purchase price paid by PURCHASER for the product This warranty shall not be effective if PLATYPUS determines in its sole discretion that PURCHASER has altered or misused the goods or has failed to use or store them in accordance with instructions furnished by PLATYPUS PLATYPUS s sole and exclusive liability and PURCHASER s exclusive remedy with respect to goods proved to PLATYPUS s satisfaction applying analytical methods reasonably selected by PLATYPUS to be defective or nonconforming shall be the replacement of such goods free of charge upon the return of such goods in accordance with our instructions although at its discretion PLATYPUS may provide a credit or refund If PLATYPUS manufactures custom goods for PURCHASER based on instructions specifications or other directions provided by PURCHASER PLATYPUS shall not be liable for the lack of sufficiency fitness for purpose or quality of the goods to the extent attributable to such instructions specifications or other directions PLATYPUS shall not be liable for any loss damage or penalty as a result of any delay in or failure to manufacture deliver or otherwise perform hereunder due to any c
7. agen Matrix solution with the cells Ye NOTE Supplements such as growth factors may be mixed with the Collagen I Matrix solution 9 Pipette a total 40 uL of the Collagen I Matrix into each test well through the side ports of the Oris Cell Seeding Stopper It is recommended to pipette 20 uL down each side port to ensure even collagen distribution throughout the well plate surface coating when introducing the pipette tip into the well Keep the pipette tips vertical while dispensing to avoid the Collagen Matrix solution getting caught on the well wall instead of the well bottom An elongated tip may be useful 10 Incubate the plate containing the Oris Cell Seeding Stoppers and Collagen Matrix in a humidified chamber 37 C 5 CO2 for 1 hour to promote collagen polymerization 11 Remove plate from incubator 12 Using the Oris Stopper Tool remove stoppers see Figure 5 Leave any stoppers in place that have been designated as pre invasion t 0 control until completion of the assay e Secure the 96 well plate by holding it firmly against the deck of your work space Slide the tines of the Oris Stopper Tool under the Figure 5 Removal of Stoppers Panels A B and C backbone of the stopper strip keeping the underside of the Oris Position the Tines of the Stopper Tool Stopper Tool flush with the top surface of the plate between the Stopper Tips D Lift Vertically and E Do NOT Pry Stoppers e Lift the Ori
8. ause beyond PLATYPUS s reasonable control PLATYPUS shall not be liable for injury or damages resulting from the use or misuse of any of its products Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 9 APPENDIX I Determining Optimal Cell Seeding Concentration Optimal cell seeding density can vary depending on the properties of each cell line being tested A suggested final concentration range is between 5 0 x 10 9 0 x 10 cell mL i e 20 000 36 000 cell well It may be helpful to evaluate various cell densities The following is an example of an experimental set up to determine optimal seeding concentration Soo Prepare a log phase culture of the cell line to be tested Collect cells and determine the total number of cells present Pellet cells by centrifugation Prepare the cell suspensions in a similar manner as described in Section IV Step 7 Continue with the assay in accordance with the remaining steps of the protocol in Section IV Dispense the collagen cell suspension into the test wells of the 96 well plate to result in the following plate layout Cells well 80 000 40 000 20 000 Number of wells 8 BT 8 amp 8 Incubate the plate in a humidified chamber 37 C 5 CO2 for 1 hour to polymerize collagen cell solution Remove the Oris Cell Seeding Stoppers from each well see Figure 5
9. ay be difficult to see the detection zone The Oris Cell Seeding Stoppers position the detection zone in the center of every well It may be helpful to use a slender pipette tip when pipetting the Collagen Matrix solution to avoid damaging to the outer collagen ring Incubate plate in a humidified chamber 37 C 5 COz for 1 hour to permit polymerization of the Collagen Matrix in the detection zone Remove plate from the incubator Add 100 uL of cell culture growth medium on top of the 3D Embedded Collagen Matrix Optional Invasion inhibitors or stimulants may be added to the medium IMPORTANT Use caution when adding media to not disturb Collagen Matrix solution Incubate plate in a humidified chamber 37 C 5 CO2 to permit cell movement the period of incubation is cell line dependent Monitor the cells periodically to check for extent of migration Refresh media or supplements every 48 72 hours as needed for the duration of the experiment If performing an endpoint analysis of cell invasion fix and stain cells with a fluorescent stain after sufficient invasion has occurred Refer to Section V and Appendices II amp Ill for further information on data acquisition and fluorescence staining technique NOTE Oris Cell Seeding Stoppers are for single use only Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypu
10. ixative solution to have a final well concentration of 0 25 glutaraldehyde solution in 1X PBS prepared from stock 8 glutaraldehyde solution Electron Microscopy Sciences Remove medium and rinse wells with 100 uL of 1X PBS Remove PBS and add 100 uL of a fixative solution final well concentration of 0 25 glutaraldehyde solution in PBS to each well and incubate at room temperature for 15 minutes Remove fixative solution and rinse wells with 100 uL of PBS Remove PBS and replace with 100 uL of a 1 50 1 100 dilution of TRITC phalloidin Sigma prepared as 10 uM stock in methanol in PBS containing 0 1 Triton X 100 Incubate plate at room temperature for 45 minutes protected from light Remove the TRITC phalloidin and add 100 uL of a 1 1000 dilution of DAPI ThermoScientific in PBS Incubate plate at room temperature for 1 hour protect from light Periodically check progress of stain Remove DAPI stain and wash wells 2x for 5 minutes each with 200 uL of PBS Replace final wash with 200 uL of fresh PBS NOTE This protocol outlines double labeling of cells with a cytoskeletal and a nuclear stain The protocol can be simplified to use only one stain Substitutions or additional cytostaining or immunostaining may be performed using non overlapping fluorophores and by utilizing the appropriate filters with your imaging equipment Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0232 01 Madison WI 53711 USA Ph
11. l the suspension concentration should be 3 75 x 10 cells mL First Time Users The optimum seeding density of cells should be determined for each cell type Please refer to Appendix for a discussion of this process Figure 4 Media is added with Single or Multi Channel Pipette IMPORTANT For recommendations on designating reference wells please refer to Section Ill Precautions and Recommendations 8 Prepare a 5 0 mL of the Collagen I Matrix solution with embedded cells using the following components e 10X PBS sterile to achieve a 1X PBS final concentration 7 5 Sodium Bicarbonate sterile 0 0125 mL mL of Oris Collagen Rat tail stock reagent Deionized water sterile Oris Collagen Rat tail Cell suspension 20 of total solution volume IMPORTANT Prior to during use keep the Oris Collagen I Rat tail and the resulting Collagen I Matrix solution on ice In addition the use of chilled pipette tips reservoirs might be beneficial The following are volumes for preparing 5 0 mL of 3 0 mg mL Collagen Matrix solution 0 5 mL 10X PBS buffer 0 0375 mL 7 5 sodium bicarbonate 0 4625 mL deionized water 3 mL Oris Collagen Rat tail 1 mL cell suspension 5 0 mL total volume On ice combine the 10X PBS 7 5 Sodium Bicarbonate and deionized water Next add the Oris Collagen Rat tail Lastly add the prepared cell suspension Gently pipette up and down to completely mix the Coll
12. late reader using appropriate filter set and sensitivity gain settings i Using the bottom probe of a fluorescence microplate reader obtain the fluorescence reading from each well To achieve the optimal dynamic range adjust the instrument settings to result in the greatest difference in fluorescence signal between pre invasion and post invasion wells Refer to the instrument manual for your microplate reader for guidance on instrument settings Additionally the plate may be analyzed by microscopy using fluorescent filters with excitation and emission wavelengths of 495 515 nm respectively Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 10 APPENDIX Ill Fluorescent Labeling Fixed Cell Options This procedure is intended to assist in obtaining data from the Oris 3D Embedded Invasion Assay using various fluorescent labels It is important to use a fluorescent reagent that uniformly stains cells Please consult the manufacturer of your fluorescent stain for specific considerations ais Q7 NOTE Use caution when adding removing solutions so that the Collagen Matrix is not dislodged from the bottom sides of 7s the well The following is an example protocol to label fixed cells with TRITC phalloidin F actin and DAPI nuclei fluorescent stains To fix one fully seeded 96 well plate prepare 10 mL of f
13. ly and are not to be used for any other purposes including but not limited to unauthorized commercial purposes in vitro diagnostic purposes ex vivo or in vivo therapeutic purposes investigational use in foods drugs devices or cosmetics of any kind or for consumption by or use in connection with or administration or application to humans or animals PLATYPUS warrants that its products shall conform substantially to the description of such goods as provided in product catalogues and literature accompanying the goods until their respective expiration dates or if no expiration date is provided for 6 months from the date of receipt of such goods PLATYPUS will replace free of charge any product that does not conform to the specifications This warranty limits PLATYPUS s liability only to the replacement of the nonconforming product THIS WARRANTY IS EXCLUSIVE AND PLATYPUS MAKES NO OTHER WARRANTY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE The stated express warranties and the remedy provided for breach thereof are in lieu of all other liability or obligations of PLATYPUS for any damages whatsoever arising out of or in connection with the delivery use misuse performance or the inability to use any of its products INNO EVENT SHALL PLATYPUS BE LIABLE UNDER ANY LEGAL THEORY INCLUDING BUT NOT LIMITED TO CONTRACT NEGLIGENCE STRICT LIABILITY IN TORT OR WARRANTY OF AN
14. m For technical assistance contact Technical Support at 866 296 4455 or techsupport platypustech com Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 ee SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 8 VIII TERMS amp CONDITIONS Certain uses of these products may be covered by U S Pat No 7 018 838 No 7 842 499 No 8 268 614 and their corresponding international applications and US and international Appl Nos 10 579 118 and 12 195 001 issued to or applied for by PLATYPUS Certain applications of PLATYPUS products may require licenses from other parties Determining the existence and scope of such third party intellectual property is the responsibility of the PURCHASER Purchase of the product provides the PURCHASER with a limited non transferable license under any PLATYPUS patents or patent applications to use the product for internal research unless there is a written limitation to this license in the product literature PURCHASER is responsible for carefully reviewing the product literature and respecting any limitations to this license e g limitations for commercial use or research by for profit institutions These products may not be resold modified for resale used to manufacture commercial products or used to develop commercial products without the express written approval of PLATYPUS These products are intended for research or laboratory use on
15. m the wells and the detection zone was filled with 3 mg mL Collagen Matrix Collagen in the detection zone was allowed to polymerize for 1 hour Complete medium 10 FBS with either GM6001 MMP inhibitor or DMSO Control was added on top of the Collagen Matrix The plate was then incubated for seven days to permit cell invasion Cells were labeled with TRITC phalloidin F actin and DAPI nuclei fluorescent stains Images were captured using a Zeiss Axio Observer Z1 inverted microscope Cells in the detection zone were quantified using ImageJ software NIH The images below illustrate representative data from full drug inhibition with 250 uM GM6001 image 1a and post invasion 0 25 DMSO image 1b wells The histogram depicts the average cell count mean SD of seven wells for number of cells invaded into the detection zones for each condition HT 1080 Cells in Oris 3D Embedded Invasion Assay 1a 1b 2 O _ O b 2 E 250 uM GM6001 0 25 DMSO 250 uM GM6001 0 25 DMSO Figure 6 Cell invasion data obtained via microscopy analysis Representative images were taken at 5X magnification with the DAPI filter set Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 hie SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 7 VI Oris PLATE DIMENSIONS Diameter of Stopper Space Detection Zone Suggested Media Volume per Well populated wi
16. one 608 237 1270 www platypustech com Fax 608 237 1271 pg 11
17. re 3 e Align the holes in the attachment lugs with the bosses on the bottom of the plate e Gently press the mask until it is flush with the bottom of the 96 well plate es e2eeeoeoeoee PS eo ee e ees e s r Pa Pa J ra T rr Ooee e2eo oe OeSE r i a K tg O7 NOTE You may wash the mask with ethanol as the mask is not sterile The mask may be applied at any point during the assay For real time assays it is most convenient to apply the mask at the beginning of the assay before any liquids are placed in the well For endpoint assays using fixed and stained cells it is most convenient to apply the mask just before reading assay results a Chamfer Attachment Lugs Figure 3 Features of Detection Mask m Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 h SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 4 Cy NOTE Do not disturb the Oris Cell Seeding Stopper or the Collagen 6 If performing real time analysis of cell invasion pre label cells with a fluorescent stain at this time Refer to Section V and Appendix Il for further suggestions on data acquisition and fluorescent labeling of live cells 7 Collect cells and prepare a suspension in cell culture medium that is 125 times greater in cell density than the target seeding concentration For example for a final concentration of 30 000 cells wel
18. s Stopper Tool vertically and remove stoppers gently s Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 h SP0232 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 5 Do not use the Oris Stopper Tool as a lever to pry the stoppers from the well see Figure 5E as doing so may cause displacement of the collagen and may distort the detection zone area NOTE You may wash the Oris Stopper Tool with 70 ethanol as the Stopper Tool is not sterile Prepare 1 5 mL of the Oris Collagen Matrix solution in a similar way as Step 8 to fill the newly created detection zone This preparation process can be started prior to step 9 immediately before the plate is removed from incubation The following are volumes for preparing 1 5 mL of 3 0 mg mL Collagen I Matrix solution 0 150 mL 10X PBS buffer 0 01125 mL 7 5 sodium bicarbonate 0 13875 mL deionized water 0 9 mL Oris Collagen Rat tail 0 3 mL media 1 5 mL total volume NOTE Supplements such as growth factors may be mixed with the Collagen I Matrix solution Pipette 10 uL of the Collagen I Matrix solution into the detection zone in the center of each well Try to get the Collagen Matrix solution all the way to the bottom of the well of the detection zone so that the material fills the cylindrical detection zone completely from bottom to top NOTE Due to the clarity of the collagen it m
19. stech com Fax 608 237 1271 pg 6 DATA ACQUISITION The readout of the Oris 3D Embedded Invasion Assay can be conducted at any time throughout as well as at the end of the assay You may use any commercially available stain or labeling technique The readout can be performed using a microscope a microplate reader or a High Content Screening or High Content Imaging instrument Microscope Analysis e Visual cell counting or image analysis software such as NIH ImageJ freeware can be used e Microscopic observations are possible using phase contrast or fluorescence microscopy e No need to attach the Oris Detection Mask to the Oris microplate e To set up reference controls refer to Section III Precautions and Recommendations Microplate Reader Analysis e Attach the Oris Detection Mask to the bottom of the Oris microplate see Section IV Step 5 e Optimal settings will vary according to the microplate reader make and model Consult Appendix II and the equipment user manual for your particular instrument e The microplate reader MUST be set to read from the bottom of the plate e To setup reference controls refer to Section Ill Precautions and Recommendations Sample Data Obtained via Microscopy are shown in Figure 6 e Oris Collagen Matrix solution at 3 mg mL with 30 000 HT 1080 cells well i e 40 uL with 3 75x10 cells mL was added to the plate and incubated 37 C 5 COz for 1 hour The stoppers were removed fro
20. th Stoppers Effective Area of Outer Annular Region seeding region per Well Plate Height Plate Height with Lid with Oris Cell Seeding Stoppers Offset of Wells A 1 location X Vil ORDERING INFORMATION 1 pack EIA1 Oris Cell Seeding Stoppers Oris 3D Embedded 3 pack EIA3 pre populated i Collagen Coated nvasion Assays Starter pack EIAST Oris Cell Seeding Stoppers p not pre populated 1 pack CMA1 101 Tissue Culture Treated 5 pack CMA5 101 Collagen Coated ee Oris Cell Migration o pack CMACC5 101 Oris Cell Seeding Stoppers Assays 1 pack CMAFN1 101 pre populated Fibronectin Coated 5 pack CMAFN5 101 TriCoated 1 pack CMATR1 101 TC Col HFn 5 pack CMATR5 101 Oris Cell Migration Tissue Culture Treated 5 pack CMAU505 Oris Cell Seeding Stoppers Assembly Kits FLEX not pre populated Tissue Culture Treated pack MAE EA 1 pack PROCMA1 Tissue Culture Treated 5 pack P Oris Pro Cell Migration Assays E ae pack PROCMACC1 Biocompatible Gel ROA 5 pack PROCMACCS 1 pack PRO384CMA1 Oris Pro 384 Tissue Culture Treated 5 pack Cee aes Cell Migration Assays Salaam ai 1 pack PRO384CMACC1 g 5 pack PRO384CMACC5 T pack PROIAT Oris Pro 3 pack PROIA3 Invasion Assays Collagen Coated pack PROIAPLUS1 3 pack PROIAPLUS3 Biocompatible Gel Biocompatible Gel For a complete list of assays visit Platyous Technologies at www platypustech co
21. to Embedded Invasion Assay Kit Cat No EIA1 is Wells C Proper Placement of Stoppers D Pressing of recommended Stoppers into Wells and E Fully Inserted Stoppers 2 4 Visually inspect the underside of the populated 96 well plate to ensure that the Oris Cell Seeding Stoppers are firmly sealed against the bottom of the plate To inspect the stoppers turn the plate over and examine the stoppers for sealing see Figure 2 If incomplete sealing is observed return the plate to the upright position and use a sterile instrument to gently push the stopper back into the well until sealing is observed SrA O NOTE The sealing of the stoppers can be most easily observed if the plate is tipped atanangle and viewed under indirect light to reveal the bullseye pattern at the bottom of each well Figure 2 5 If microplate reader data will be collected apply the Oris Detection Mask to the Figure 2 Stoppers that are bottom of the 96 well plate The Detection Mask is not necessary if collecting imaging i Pori data C Completely Sealed First Time Users In order to prevent splashing of well contents familiarize yourself with the attachment and removal of the Detection Mask before any liquids are placed into the wells Aperture Orientation A 1 Corner e Orient the chamfered corners of the mask with those of the 96 well plate ensuring that the A1 corner of the mask is aligned with the A1 well of the plate Figu
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