Oris™ Cell Migration Assay - Fibronectin
Contents
1. gt L AT Y PUS Bringing Science to the Surface Ons Cell Migration Assay Fibronectin Coated Product No CMAFN1 101 amp CMAFNS5 101 96 well 2 D Assay for Investigating Cell Migration of Adherent Cell Lines on Fibronectin PROTOCOL amp INSTRUCTIONS Table of Contents INTRODUCTION ORIS PLATE DIMENSIONS MATERIALS PROVIDED MATERIALS REQUIRED CELL MIGRATION ASSAY FIBRONECTIN COATED PROTOCOL DATA ACQUISITION ORDERING INFORMATION TERMS amp CONDITIONS Appendix Determining Optimal Cell Seeding Concentration Appendix Il Determining Optimal Fluorescence Microplate Reader Settings PLATYPUS 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 SP0032 03 Oris CELL MIGRATION ASSAY FIBRONECTIN COATED I INTRODUCTION The Oris Cell Migration Assay Fibronectin Coated is a reproducible sensitive and flexible assay that can be used to monitor cell migration 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 the 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 visua2. Analysis No Mask Required Figure 1 Schematic of Oris Cell Migration Assay Fibronectin Coated Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0032 03 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 2 ll ORIS PLATE DIMENSIONS Effective Area of Outer Annular Region seeding region per Well 30 03 mm Plate Height with Lid with Oris Cell Seeding Stoppers 17 9 mm Offset of Wells A 1 location X 14 4 mm Offset of Wells A 1 location Y 11 2 mm Storage Conditions Refrigerate 4 C Important Read Instructions Before Performing any Oris Assay lll MATERIALS PROVIDED Product No CMAFN1 101 Product No CMAFN5 101 Oris Fibronectin Coated 96 well black clear bottom Oris Fibronectin Coated 96 well black clear bottom Plate with Oris Cell Seeding Stoppers 1 Plates with Oris Cell Seeding Stoppers 5 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 CellTr
3. 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 Fibronectin Coated 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 culture 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 tested Collect cells and determine the total number of cells present Pellet cells by centrifugation Prepare three cell suspensions in culture media at final concentrations of 1 0 x 10 0 5 x 10 and 0 25 x 10 cells mL Dispense 100 uL of cell suspension per well into the 96 well plate to result in the following plate layout Cells well 100 000 50 000 25 000 Number of wells 8 8 8 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 Fibronectin coated surfa
4. 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 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 SP0032 02 Madison
5. migration assay per Section V Cell Migration Assay Fibronectin Coated 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 Perform the desired fluorescent staining technique The Oris Cell Migration Assay Fibronectin Coated 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 experimental 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 Tostain 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 solution from light until ready to use Carefully remove culture medium from wells Wash wells with 100 uL of PBS containing both Ca and Mg Add 100 uL of di
6. 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 refer to Section V step 3 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 The microplate reader MUST be set to read from the bottom of the plate Sample data using a fluorescent stain and microplate reader analysis are shown in Figure 6 Fibronectin coated wells populated with Oris Cell Seeding Stoppers were seeded with 35 000 HT 1080 cells well i e 100 uL at 3 5 x 10 cells mL The plate was incubated for 8 hours at 37 C 5 CO2 The stoppers were then removed from test wells Stoppers were left in place in reference wells until the staining step to serve as pre migration controls After an additional 16 hours to allow for migration cells were stained with Calcein AM for 30 minutes The relative fluorescence units RFU were then measured using a microplate reader with the Detection Mask in place The graph Figure 6B depicts the fluorescence signal present in the detection zones for each condition mean S D n 32 wells condition The images below Figure 6A captured without a Detection Mask in place illustrate representative data from pre migration reference wells and post migration test wells 6B HT 1080 Cell Migrati
7. to Appendix for a discussion of this process Pipette 100 uL of suspended cells into each test well through one of the side ports of the Oris Cell Seeding Stopper Figure 2 Stoppers that are A Partially Sealed B Unsealed C Completely Sealed Aperture Orientation A 1 Corner A j eeeocece r E E ec 6 aT oe ee ee a ee o nm 2 r eoeoeeeec amp a 2 amp a oo Ege gt eeeeoenceee e e ie a ia w a o Chamfer Attachment Lugs Figure 3 Features of Detection Mask Figure 4 Media is Added with Single or Multi Channel Pipette NOTE For best results add or extract media by placing the pipette tip along the wall of the well see Figure 4 Care should be taken not to disturb the Fibronectin Coating or the Oris Cell Seeding Stopper when introducing the pipette tip into the well A slender elongated tip or a gel loading tip may be useful IMPORTANT Lightly tap the plate on your work surface to evenly distribute well contents extreme tapping may result in splashing of well contents and lead to contamination 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 to permit cell attachment eee Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0032 02 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg
8. 4 V CELL MIGRATION ASSAY FIBRONECTIN COATED PROTOCOL continued 9 Remove plate from incubator 10 Designate several reference wells in which the stoppers will remain in place until results are read t O pre migration controls 11 Using the Oris Stopper Tool remove all other stoppers see Figure 5 127 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 lr YY NOTE DO NOT use the Oris Stopper Tool as a lever to pry E the stoppers from the well see Figure 5E as doing so may cause displacement of seeded cells and may distort the detection zone area 12 Remove media with a pipette and gently wash wells with 100 uL of sterile PBS or media to remove any unattached cells Do not aspirate using an in house vacuum 13 Add 100 uL of fresh culture media to each well seus Figure 5 Removal of Stoppers Panels A B and C Position 14 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 Ve
9. acker 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 s Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0032 02 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 3 PLATYPUS CELL MIGRATION ASSAY FIBRONECTIN COATED PROTOCOL The following steps should be performed in a biological hood using aseptic technique to prevent contamination 1 Tr p1 Remove the Oris Fibronectin Coated Plate with Cell Seeding Stoppers from refrigeration and place on lab bench for 1 hour to allow it to equilibrate to room temperature 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 NOTE The sealing of the stoppers can be most easily observed if the plate is tipped at an angle and viewed under indirect light to reveal the bullseye pattern at the bottom of each well Apply the Oris Detection Mask to the bottom of the 96 wel
10. ce Following cell attachment remove the Oris Cell Seeding Stoppers from each well see Figure 5 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 Stopper Tool under the backbone of the stopper strip keeping the underside of the Stopper Tool flush with the top surface of the plate e Lift the Stopper Tool vertically to gently remove the stopper Do not use the Stopper Tool as a lever to pry the stoppers from the well as doing so may cause displacement of the seeded cells 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 perimeter of the detection zone At this point if you plan to obtain the results of the Oris Cell Migration Assay Fibronectin Coated via colorimetric or microscopy analysis you have successfully determined the cell seeding concentration to be used at Step 5 of the Cell Migration Assay Fibronectin Coated 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 Fibronectin Coated 1 Using the optimal cell seeding concentration determined in Appendix perform a cell
11. l plate if microplate reader data is being collected The Detection Mask is not necessary if collecting imaging data 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 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 see Figure 3 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 NOTE It may be necessary to wash the mask with ethanol to remove dust and 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 If performing a kinetic analysis of cell migration pre label cells with a fluorescent stain now 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
12. lization to the detection zones allowing only cells that have migrated to be detected see Figure 1 The Oris Cell Migration Assay Fibronectin Coated 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 Fibronectin Coated 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 and NMuMG cell lines Using the Oris Cell Migration Assay Fibronectin Coated 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 Specific measure cell migration directly on an extracellular matrix coated surface Seed amp Adhere Cells Remove Allow Cells to Analyze Cells in Detection onto Oris Fibronectin Stoppers to Migrate into Zone Microplate Reader Coated Plate Create Detection Detection Zone Analysis Detection Mask Zone Attached Image
13. luted Calcein AM Reagent 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 Biolek Synergy HT microplate reader use 485 528 nm excitation emission filters sensitivity 55 nm 7 DQ200 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 You have now successfully determined the optimal cell seeding concentration to be used in Step 5 of the Cell Migration Assay Fibronectin Coated Protocol and microplate reader settings for analysis of cell migration using a fluorescence microplate reader PLATYPUS Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0032 02 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 8
14. nologies at www platypustech com order_main html For technical assistance contact Technical Support at 866 296 4455 or techsupport platypustech com VIII 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 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 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 f
15. on into Detection Zone Mean Cell Migration RFU s Post Migration Well Pre migration Post Migration Figure 6 Cell migration data obtained using Calcein AM fluorescent stain Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0032 02 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 6 PLATYPUS VII ORDERING INFORMATION 1 pack PROCMA1 eee Tissue Culture Treated 5 pack PROCMAS ae Biocompatible Gel Cell Migration Assays E EEE Cantal 1 pack PROCMACC1 amp 5 pack PROCMACCS Tissue Culture Treated 5 pack PRO384CMAS Oris Pro 384 ae Biocompatible Gel Cell Migration Assays Collagen I Coated 5 pack PRO384CMACC35 1 pack CMA1 101 5 pack CMA5 101 Tissue Culture Treated Collagen I Coated I pack CMACC1 101 Oris Cell Migration 5 pack CMACC5 101 Onis Celi S ading Stoppers Assays pre populated Fibronectin Coated ee 5 pack CMAFNS 101 I pack CMATR1 101 Universal 1 pack CMAU101 Oris Cell Migration Tissue Culture Treated 5 pack CMAUS05 Oris Cell Seeding Stoppers Assembly Kits not pre populated FLEX Tissue Culture Treated 4 pack CMAUFL4 Collagen I 1 pack PROIA1 l Oris Pro 96 well low overlay conc 3 pack PROIA3 Ee Invasion Assays Collagen I 1 pack PROIAPLUS1 l high overlay conc 3 pack PROIAPLUS3 Biocompanbie el For a complete list of assays visit Platypus Tech
16. or 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 IN NO 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
17. rtically 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 plate coating 15 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 APA NOTE Oris Cell Seeding Stoppers are for single use only Platypus cannot guarantee the integrity of the stopper material after a second sterilization procedure Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 HS sP0032 02 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 Fibronectin Coated can be conducted at any time allowing the user to perform a kinetic assay or an endpoint assay The Oris Cell Migration Assay Fibronectin Coated 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 Microscopy 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
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