Oris™ Cell Migration Assay - TriCoated Protocol
Contents
1. Cell Seeding Stoppers Assays re populated l pack CMAFN1 101 ERAR 5 pack CMAFNS 101 1 pack CMATR1 101 Universal l pack CMAU101 Oris Cell Migration Tissue Culture Treated 5 pack CMAUS05 Oris Cell Seeding Stoppers Assembly Kits not pre populated Collagen I Coated Fibronectin Coated FLEX Tissue Culture Treated 4 pack CMAUFL4 Collagen I 1 pack PROIA1 Oris Pro 96 well low overlay conc 3 pack PROIA3 et kee Invasion Assays Collagen I 1 pack PROIAPLUS1 l high overlay conc 3 pack PROIAPLUS3 Biocompare For a complete list of assays visit Platyous Technologies at www platypustech com order_main html For technical assistance contact Technical Support at 866 296 4455 or techsupport platypustech com Vill TERMS amp CONDITIONS PLATYPUS Certain uses of these products may be covered by U S Pat No 7 842 499 issued to or patents 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 re2. You have now successfully determined the optimal cell seeding concentration to be used in Step 5 of the Cell Migration Assay TriCoated Protocol and microplate reader settings for analysis of cell migration using a fluorescence microplate reader T Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0034 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 8
3. 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 ANY 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 P
4. of well contents and lead to contamination 8 Incubate the seeded plate containing the Oris Cell Seeding Stoppers in a humidified chamber 37 C 5 CO2 for 4 to 18 hours cell line dependent and surface treatment dependent to permit cell attachment T Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0034 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 4 CELL MIGRATION ASSAY TRICOATED PROTOCOL continued Remove media with a pipette and gently wash wells with Add 100 uL of fresh culture media to each well Remove plate from incubator Designate several reference wells for each surface treatment in which the stoppers will remain in place until results are read t 0 pre migration controls Using the Oris Stopper Tool remove all other stoppers see Figure 6 NOTE It may be necessary to wash the Oris Stopper Tool with 70 ethanol as the Stopper Tool is not sterile 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 backbone of the stopper strip keeping the underside of the Oris Stopper Tool flush with the top surface of the plate e Lift the Oris Stopper Tool vertically to gently remove the stoppers NOTE DO NOT use the Oris Stopper Tool as a lever to pry the stoppers from the well see Figure 6E as doing so may cause displacemen
5. PLAT YRU2 Bringing Science to the Surface Ons Cell Migration Assay TiCoated Product No CMATR1 amp CMATRS5 96 well 2 D Assay for Investigating Cell Migration of Adherent Cell Lines on Three Coated Surfaces PROTOCOL amp INSTRUCTIONS Table of Contents INTRODUCTION ORIS PLATE DIMENSIONS MATERIALS PROVIDED MATERIALS REQUIRED CELL MIGRATION ASSAY TRICOATED PROTOCOL DATA ACQUISITION ORDERING INFORMATION TERMS amp CONDITIONS Appendix Determining Optimal Cell Seeding Concentration Appendix Il Determining Optimal Fluorescence Microplate Reader Settings 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 SP0034 03 Oris CELL MIGRATION ASSAY TRICOATED INTRODUCTION The Oris Cell Migration Assay TriCoated is a reproducible sensitive and flexible assay that can be used to monitor cell migration on three differently coated surfaces Optimizing conditions for conducting cell migration studies can be costly and time consuming The new Oris Cell Migration Assay TriCoated combines three kits into one for evaluating the best surface coating for your cell migration application Formatted for a 96 well plate the assay utilizes Oris Cell Seeding Stoppers made from a medical grade silicone to restrict cell seeding to the outer annular regions of the wells Removal of t
6. URCHASER 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 cause 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 SP0034 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 7 APPENDIX I Determining Optimal Cell Seeding Concentration This procedure is intended to assist in determining the cell seeding density needed to achieve confluency of your cell line when using the Oris Cell Migration Assay TriCoated The intended goal is to achieve 90 95 confluency of the monolayer surrounding the Oris Cell Seeding Stoppers without overgrowth 1 A suggested starting point is to evaluate three serial dilutions at the cell densities shown below The cell seeding area of the well with the stopper in place is 0 3 cm Based on the typical seeding density of your particular cell line you can infer a different cell number for your first serial dilution and adjust the numbers below accordingly Prepare a log phase culture of the cell line to be tes
7. al conditions will increase variability of results and reduce correlation between fluorescence signal and cell migration Please consult the manufacturer of your fluorescent stain for specific considerations The following is an example Fluorescent Staining Protocol for using Calcein AM a To stain one fully seeded 96 well plate combine 5 uL of Calcein AM 1 mg mL in dry DMSO with 10 mL of phenol red free and serum free media or 1x PBS containing both Ca and Mg Protect diluted Calcein AM solution from light until ready to use in step d Carefully remove culture medium from wells 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 microplate reader using appropriate filter set and sensitivity gain settings for a BioTek Synergy HT microplate reader use 485 528 nm excitation emission filters sensitivity 55 nm oOo 3 If not already in place apply the Oris Detection Mask to the plate 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 e g gain to result in the greatest difference in fluorescence signal between pre migration and post migration wells Refer to the instrument manual for your microplate reader for further guidance on instrument settings
8. erimeter of the detection zone At this point if you plan to obtain the results of the Oris Cell Migration Assay TriCoated via colorimetric or microscopic analysis you have successfully determined the optimal cell seeding concentration to be used in Step 5 of the Cell Migration Assay TriCoated Protocol APPENDIX II Determining Optimal Fluorescence Microplate Reader Settings This procedure is intended to assist in optimizing your instrument settings when using a fluorescence microplate reader to capture data from the Oris Cell Migration Assay TriCoated 1 Using the optimal cell seeding concentration determined in Appendix perform a cell migration assay per Section V Cell Migration Assay TriCoated Protocol using culture conditions expected to result in robust cell migration Be sure to include equal numbers of pre migration reference wells stoppers left in place until staining and post migration test wells stoppers removed after cell attachment period A minimum of 8 wells per condition are recommended 2 Perform the desired fluorescent staining technique The Oris Cell Migration Assay TriCoated has been designed to work with all types of fluorescent stains and staining techniques The precise method for staining cells with fluorescent stains varies according to the nature of the individual stain It is important to stain cells using a fluorescent reagent that uniformly stains cells Probes affected by experiment
9. form a kinetic assay or an endpoint assay The Oris Cell Migration Assay TriCoated is designed to be used with any commercially available stain or labeling technique The readout can be performed by using a microscope a microplate reader or a High Content Screening or High Content Imaging Analysis platform Microscope Analysis e Cell counting or image capture analysis software such as NIH ImageJ freeware can be used e Note Microscopy observations are possible using phase contrast or bright field microscopy e No need to attach the Oris Detection Mask to the Oris plate Microplate Reader Analysis e Attach the Oris Detection Mask to the bottom of the Oris plate see Step 3 of Protocol 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 Sample data using a fluorescent stain and microplate reader analysis are shown in Figure 7 Tissue Culture Treated Collagen Coated and Fibronectin Coated wells populated with Oris Cell Seeding Stoppers were seeded with 25 000 NMuMG cells well i e 100 uL at 2 5 x 10 cells mL The plate was incubated for 7 hours at 37 C 5 CO2 The stoppers were then removed from test wells Stoppers were left in place in reference wells n 8 surface treatment until the staining step to serve as pre migratio
10. h the mask with ethanol to remove dust and E debris since the mask is not sterile The mask may be applied at any point during the assay For kinetic assays it is often 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 often most convenient to apply the mask just before reading assay results 4 f performing a kinetic analysis of cell migration pre label cells with a fluorescent stain now 5 Collect cells and prepare a suspension that is 10 fold greater in density than the optimal seeding concentration First Time Users The optimum seeding density of cells must be determined as an integral part of the design of the cell migration assay Please refer to Appendix for a discussion of this process Figure 5 Media is Added with Single or Multi Channel Pipette 6 Pipette 100 uL of suspended cells into each test well through one of the side ports of the Oris Cell Seeding Stopper A O NOTE For best results add or extract media by placing the pipette tip along the wall of the well see Figure 5 Care should g be taken not to disturb the well coatings or the Oris Cell Seeding Stoppers when introducing the pipette tip into the well A slender elongated tip or a gel loading tip may be useful 7 IMPORTANT Lightly tap the plate on your work surface to evenly distribute well contents extreme tapping may result in splashing
11. he stoppers reveals a 2mm diameter unseeded region in the center of each well i e the detection zone into which the seeded cells may then migrate The Oris Detection Mask is applied to the plate bottom and restricts visualization to the detection zones allowing only cells that have migrated to be detected see Figure 1 The Oris Cell Migration Assay TriCoated provides wells that are Tissue Culture Treated Collagen coated and Fibronectin coated see Figure 2 The kit is designed to be used with any commercially available stain or labeling technique Readout can be performed by microscopy or use of a microplate reader The Oris Cell Migration Assay TriCoated system has been designed for use with adherent cell cultures This assay has been successfully used with HT 1080 PC 3 A549 NCI H1650 MDA MB 231 HUVEC and NMuMG cell lines Using the Oris Cell Migration Assay TriCoated offers the following features amp benefits e Membrane free Migration perform studies e Versatile analyze data using multiple probes in a single without manipulating transmembrane inserts well by using a microscope digital imager or fluorescence e Reproducible Results obtain well to well CV s lt 12 microplate reader due to the unique design e Flexible perform kinetic or endpoint cell migration e Preserves Cell Morphology monitor changes in cell assays without the use of special instrumentation structure in real time e Opt
12. imize Migration compare cell migration on three differently coated surfaces all on one plate Seed amp Adhere Remove Allow Cells to Analyze Cells in Detection Cells onto Stoppers to Migrate into Zone Microplate Reader Oris Create Detection Detection Zone Analysis Detection Mask TriCoated Plate Zone Attached Image Analysis No Mask Required Figure 1 Schematic of Oris Cell Migration Assay TriCoated Tissue Culture Treated Collagen C Fibronectin Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0034 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 2 ll ORIS PLATE DIMENSIONS per well Diameter of Stopper Space Detection Zone Suggested Media Volume per Well populated with Stoppers Effective Area of Outer Annular Region seeding region per Well Plate Height with Lid with Oris Cell Seeding Stoppers Offset of Wells A 1 location X Offset of Wells A 1 location Y Distance between Wells 9 mm on center Thickness of Well Bottom TriCoated Plate Contents see Figure 2 aa Important Read Instructions Before Performing any Oris Assay ll MATERIALS PROVIDED Product No CMATR1 101 Product No CMATR5 101 Oris TriCoated 96 well Plate with Oris Cell Seeding Oris TriCoated 96 well Plates with Oris Cell Stoppers Seeding Stoppers 5 32 wells Tissue Culture Treated 32 wells Tissue Culture Treated 32 we
13. lls Collagen coated 32 wells Collagen coated 32 wells Fibronectin coated 32 wells Fibronectin coated Oris Detection Mask 1 Oris Detection Mask 1 Oris Stopper Tool 1 Oris Stopper Tool 1 IV MATERIALS REQUIRED Biological Cells Sterile PBS containing both Calcium and Magnesium Complete Cell Culture Growth Medium containing serum Sterile Pipette Tips Pipette or Multi Channel Pipette Trypsin or Cell Scraper Inverted Microscope optional Fluorescence Microplate Reader optional Cell Culture Labeling Medium phenol red free serum free media Cell Labeling Fluorescent Agent eg CellTracker Green Calcein AM required if performing assay readout via microplate reader Oris is a trademark of Platypus Technologies LLC CellTracker Green is a trademark of Invitrogen Corporation 7 Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 E SP0034 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 3 CELL MIGRATION ASSAY TRICOATED PROTOCOL The following steps should be performed in a biological hood using aseptic technique to prevent contamination 1 Remove the Oris TriCoated Plate with Cell Seeding Stoppers from refrigeration and place on lab bench for 1 hour to allow it to equilibrate to room temperature 2 Visually inspect the underside of the populated 96 well plate to ensure that the Oris Cell Seeding S
14. n controls After an additional 16 hours to allow for migration cells were fluorescently stained with Calcein AM for 30 minutes Fluorescence was then measured using a microplate reader with the Detection Mask in place The images below 7A captured without a Detection Mask in place illustrate representative data from pre migration reference wells 0 hr and post migration test wells 16 hr The graph 7B depicts the relative fluorescence units RFU s present in the detection zones for each condition mean S D n 8 wells condition NMuMG Cell Migration into Detection Zone Tissue Culture Treated Collagen Fibronectin D LL x o D Q gt lt Tissue Culture Treated Collagen Coated Fibronectin Coated Figure 7 Cell migration data obtained using Calcein AM fluorescent stain T Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0034 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 6 VII ORDERING INFORMATION l l pack PROCMA1 E Tissue Culture Treated 5 pack PROCMAS oga D Biocompatible Gel Cell Migration Assays EET E 1 pack PROCMACC1 amp 5 pack PROCMACCS Tissue Culture Treated 5 pack PRO384CMAS Oris Pro 384 aA Biocompatible Gel Cell Migration Assays Collagen I Coated 5 pack PRO384CMACCS5 1 pack CMA1 101 Tissue Culture Treated 5 pack CMAS 101 1 pack CMACC1 101 Oris Cell Migration S pack CMACCS 101 Oris
15. specting 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 only 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
16. t of seeded cells and may distort the detection zone area 100 uL of sterile PBS or media to remove any unattached cells Do not aspirate using an in house vacuum Figure 6 Removal of Stoppers Panels A B and C Position Incubate plate in a humidified chamber 37 C 5 CO2 to the Tines of the Stopper Tool between the Stopper permit cell migration Cells may be examined microscopically Tips D Lift Vertically and E Do NOT Pry throughout the incubation period to monitor progression of Stoppers migration Migration time will vary depending upon cell type experimental design and surface treatment as different ECM s have been shown to have varying effects on migration even for a given cell line If performing an endpoint analysis of cell migration stain cells with a fluorescent stain after sufficient migration has occurred Refer to Section VI and Appendix II for further information on data acquisition and fluorescence staining technique NOTE Oris Cell Seeding Stoppers are for single use only Platypus cannot guarantee the integrity of the stopper material after a second sterilization procedure ee Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0034 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 5 VI DATA ACQUISITION The readout of the Oris Cell Migration Assay TriCoated can be conducted at any time allowing the user to per
17. ted Collect cells and determine the total number of cells present Pellet cells by centrifugation Prepare two serial dilutions at final concentrations of 0 5 x 10 and 0 25 x 10 cells mL Dispense 100 uL of cell suspension per well into the 96 well plate according to the following table 50 000 Cells Well 25 000 Cells Well Tissue Culture Treated Wells 2 A1 B1 C1 D1 aan a Collagen I Coated Wells 2 A5 B5 C5 D5 Fibronectin Coated Wells 2 A9 B9 C9 DY 5 Incubate the plate in a humidified chamber 37 C 5 CO2 for 4 18 hours cell line dependent with cell seeding stoppers in place to allow the cells to firmly attach to the well surface 6 Following cell attachment remove the Oris Cell Seeding Stoppers from each well see Figure 6 and gently wash the wells with PBS to remove non attached cells 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 backbone of the stopper strip keeping the underside of the tool flush with the top surface of the plate e Liftthe Oris Stopper Tool vertically to gently remove the stopper Do not use the Oris Stopper Tool as a lever to pry the stoppers from the well as doing so may cause displacement of the seeded cells 7 Without a Detection Mask in place use a microscope to visually inspect each well to determine the minimum cell seeding concentration that yields a confluent monolayer at the p
18. toppers 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 3 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 k NOTE The sealing of the stoppers can be most easily observed if the plate is Figure 3 e ey a j tipped at an angle and viewed under indirect light to reveal the bullseye pattern at a fe ear the bottom of each well C Completely Sealed 3 Apply the Oris Detection Mask to the bottom of the 96 well plate if microplate reader data is being collected The Detection Mask is not necessary if collecting imaging data Aperture Orientation A 1 Corner First Time Users In order to prevent splashing of well contents familiarize eeeee yourself with the attachment and removal of the Detection Mask before any liquids og are placed into the wells tv ee a w a e Orient the chamfered corners of the mask with those of the 96 well plate es ensuring that the A1 corner of the mask is aligned with the A1 well of the plate b 7 see Figure 4 ce e Align the holes in the attachment lugs with the bosses on the bottom of the 96 well plate e Gently press the mask until it is flush with the bottom of the 96 well plate Chamfer Attachment Lugs Figure 4 Features of Detection Mask O NOTE It may be necessary to was
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