OrisTM Collagen I Cell Invasion Assay
Contents
1. A suggested starting concentration for the Oris Collagen I Overlay is 3 mg mL Recommendations for Preparation of Reference Wells To establish t 0 pre invasion reference wells by seeding both test and reference wells at the same time it is necessary to seed cells at different concentrations Seed test wells at a density determined optimal in Appendix I but seed reference wells at sub optimal density 50 75 confluency Allow cells to adhere in all wells and remove stoppers from test wells treat test wells with the Oris Collagen I Overlay incubate to allow for gel formation and proceed with invasion experiment Reference wells will remain populated with Oris Cell Seeding Stoppers until the end of the assay At that point remove the Oris Cell Seeding Stoppers from the reference wells treat the wells with the Oris Collagen I Overlay incubate to allow for gel formation and proceed with staining analysis of the entire plate To establish t 0 pre invasion reference wells by seeding test and reference wells at the same concentration it is necessary to seed cells at different times during the assay Seed test wells at density determined optimal in Appendix I allow cells to adhere for 1 18 hours remove stoppers treat with the Oris Collagen I Overlay and incubate to allow for gel formation Allow cells to invade for a set amount of time At 1 18 hours prior to analyzing test wells seed reference wells at a density dete2. 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 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
3. Figure 4 Media is Added with Single or Multi Channel Pipette Orientation A 1 Corner Aperture Chamfer Attachment Lugs Figure 3 Features of Detection Mask Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 RM0039 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 6 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 8 Incubate the seeded plate containing the Oris Cell Seeding Stoppers in a humidified chamber 37 C 5 CO2 for 1 to 18 hours cell line dependent to permit cell attachment 9 Remove plate from incubator 10 Using the Oris Stopper Tool remove stoppers see Figure 5 NOTE It may be necessary to wash the Oris Stopper Tool with 70 ethanol as the Stopper Tool is not sterile 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 Lift the Oris Stopper Tool vertically to gently remove stoppers 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 seeded cells and may distort the detection zone area 11 Remove
4. PROTOCOL amp INSTRUCTIONS I INTRODUCTION 2 II ORISTM PLATE DIMENSIONS 3 III MATERIALS PROVIDED 3 IV MATERIALS REQUIRED 4 V PRECAUTIONS AND RECOMMENDATIONS 4 VI COLLAGEN I CELL INVASION ASSAY PROTOCOL 5 VII DATA ACQUISITION 7 VIII ORDERING INFORMATION 8 IX TERMS amp CONDITIONS 8 APPENDIX I Determining Optimal Cell Seeding Concentration 9 APPENDIX II Fluorescent Labeling Live Cell Options 9 APPENDIX III Fluorescent Labeling Fixed Cell Options 10 RM0039 01 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 Bringing Science to the SurfaceTM OrisTM Collagen
5. 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 Product Name Coating Size Detection Zone Format Oris Collagen I Cell Invasion Assay Collagen I 1 pack CIA101CC 2 pack CIA200CC Oris Cell Seeding Stoppers pre populated Oris Cell Invasion amp Detection Assay BME 1 pack CIA101DE 2 pack CIA200DE Oris Cell Seeding Stoppers not pre populated Oris Pro Cell Migration Assays Tissue Culture Treated 1 pack PROCMA1 5 pack PROCMA5 Biocompatible Gel Collagen I Coated 1 pack PROCMACC1 5 pack PROCMACC5 Oris Cell Migration Assays Tissue Culture Treated 1 pack CMA1 101 5 pack CMA5 101 Oris Cell Seeding Stoppers pre populated Collagen I Coated 1 pack CMACC1 101 5 pack CMACC5 101 Fibronectin Coated 1 pack CMAFN1 101 5 pack CMAFN5 101 TriCoated 1 pack CMATR1 101 5 pack CMATR5 101 Oris Cell Migration Assembly Kits Universal Tissue Culture Treated 1 pack CMAU101 5 pack CMAU505 Oris Cell Seeding Stoppers not pre populated Collagen I Coated 1 pack CMAUCC1 5 pack CMAUCC5 FLEX Tissue Culture Treated 4 pack CMAUFL4 Platypus Technologies LLC 5520 Nobel Drive Suite 100 Tol
6. media with a pipette and gently wash wells with 100 L of serum free media or sterile PBS to remove any unattached cells Do not aspirate using an in house vacuum IMPORTANT Prior to during use keep the Oris Collagen I Stock Reagent and the Oris Collagen I Overlay Solution on ice In addition the use of chilled pipette tips reservoirs might be beneficial 12 Prepare 5 0 mL of an appropriate concentration of the Oris Collagen I Overlay solution using the following components Oris 10X PBS sterile 7 5 sodium bicarbonate sterile Deionized water sterile Oris Collagen I Stock Reagent 5 mg mL Calculate the volume of Oris Collagen I Stock Reagent needed to make the desired concentration of the Oris Collagen I Overlay solution Calculate the volume of sodium bicarbonate needed to neutralize the collagen where 0 0125ml of 7 5 sodium bicarbonate is required for every 1 mL of 5 mg mL Oris Collagen I Stock Reagent used Appropriate volumes of 10X PBS and deionized water are used to prepare the Collagen I overlay in a final 1X PBS solution On ice combine the water Oris 10X PBS and sodium bicarbonate Next add the Oris Collagen I Stock Reagent to achieve the desired concentration of the Collagen I Overlay solution The following example protocol provides volumes for a 3 0 mg mL Collagen I Overlay solution 1 4625 mL deionized water 0 5 mL Oris 10X PBS buffer 0 0375 mL 7 5 sodium bicarbonate 3 mL Oris
7. 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 5 If performing kinetic analysis of cell invasion pre label cells with a fluorescent stain at this time Refer to Section VII and Appendix II for further information on data acquisition and fluorescent labeling of live cells 6 Collect cells and prepare a suspension that is 10 fold greater in density than the optimal seeding concentration using complete cell culture growth medium containing serum First Time Users The optimum seeding density of cells must be determined as an integral part of the design of the cell invasion assay Please refer to Appendix I for a discussion of this process IMPORTANT For recommendations on designating reference wells please refer to Section V Precautions and Recommendations 7 Pipette 100 L of suspended cells into each test well through one of the side ports of the Oris Cell Seeding Stopper 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 Oris Cell Seeding Stopper or the Collagen I coating when introducing the pipette tip into the well A slender elongated tip or a gel loading tip may be useful Figure 2 Stoppers that are A Partially Sealed B Unsealed C Completely Sealed A B C
8. Collagen I Stock Reagent 5mg mL 5 0 mL total volume NOTE Supplements such as growth factors may be mixed with the 3 D Collagen I Overlay 13 Remove media from wells and add 40 L of the Oris Collagen I Overlay to each well IMPORTANT Place plate on ice during addition of the Oris Collagen I Overlay to reduce premature polymerization of the Collagen I 14 Incubate plate in a humidified chamber 37 C 5 CO2 for 1 hour to permit polymerization of the 3 D Collagen I overlay 15 Add 100 L of complete media containing serum on top of the 3 D Collagen I Overlay Optional Invasion inhibitors or stimulants may be added to the media A B C D E Figure 5 Removal of Stoppers Panels A B and C Position the Tines of the Stopper Tool between the Stopper Tips D Lift Vertically and E Do NOT Pry Stoppers Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 RM0039 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 7 IMPORTANT Use caution when adding media so as not to dislodge the Collagen I Overlay from bottom sides of the well 16 Incubate plate in a humidified chamber 37 C 5 CO2 to permit cell invasion length of incubation is cell line dependent Refresh media or supplements every 48 72 hours as needed for the duration of the invasion experiment 17 If performing an endpoint analysis of cell invasion stain cells wi
9. I Cell Invasion Assay Product No CIA101CC amp CIA200CC 96 well 3 D Assay for Investigating Cell Invasion of Adherent Cell Lines on Collagen I Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 RM0039 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 2 ORIS COLLAGEN I CELL INVASION ASSAY I INTRODUCTION The Oris Collagen I Cell Invasion Assay is a reproducible sensitive and flexible assay that can be used to monitor cell invasion 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 2 mm diameter unseeded region in the center of each well i e the detection zone into which the seeded cells may then invade once the Collagen I Overlay has been applied The Oris Detection Mask is applied to the plate bottom and restricts visualization to the detection zone thus allowing only motile cells to be detected see Figure 1 The Oris Collagen I Cell Invasion Assay 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 The Oris Collagen I Cell Invasion Assay kit has been uniquely designed to detect cellular migration and
10. 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 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
11. ding 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 NOTE Use caution when adding removing solutions so that the Collagen I Overlay 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 To stain one fully seeded 96 well plate combine 5 L 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 b Carefully remove culture medium from wells c Wash wells with 100 L of PBS containing both Ca and Mg d Add 100 L of diluted Calcein AM solution to each well e Incubate plate at 37 C for 30 60 minutes f 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 If not already in place apply the Oris Detection Mask to the plate Using the bottom probe of a fluorescence microplate reader obtain the fl
12. formed using non overlapping fluorophores and by utilizing the appropriate filters with your imaging equipment At this point you have successfully fixed and labeled your cells
13. he Oris Cell Seeding Stoppers from each well see Figure 6 and gently wash the wells with PBS to remove non attached cells 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 Lift the 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 yielded a confluent monolayer at the perimeter of the detection zone At this point if you plan to obtain the results of the Oris Collagen I Cell Invasion Assay via colorimetric analysis or microscopy you have successfully determined the optimal cell seeding concentration to be used in Step 6 of the Oris Collagen I Cell Invasion Assay Protocol APPENDIX II Fluorescent Labeling Live Cell Options This procedure is intended to assist in obtaining data from the Oris Collagen I Cell Invasion Assay using various fluorescent labels The Oris Collagen I Cell Invasion Assay has been designed to work with all types of fluorescent stains and staining techniques The precise method for staining cells with fluorescent stains varies accor
14. invasion in vitro within a 3 dimensional extracellular matrix comprised of Collagen I rat tail The Oris Collagen I Cell Invasion Assay system has been designed for use with adherent cell cultures Performance of the assay was optimized using HT 1080 fibrosarcoma and MDA MB 231 breast epithelial cell lines Using the Oris Collagen I Cell Invasion Assay offers the following features amp benefits Membrane free Invasion perform studies without manipulating transmembrane inserts no membrane to restrict the ability to image cells Reproducible Results obtain low well to well CV s due to the unique assay design Preserve Cell Morphology realize a more native 3 D environment Versatile analyze data using multiple probes in a single well by using a microscope digital imager or fluorescence microplate reader Flexible perform real time or endpoint cell invasion assays without the use of special instrumentation Figure 1 Schematic of Oris Collagen I Cell Invasion Assay Remove Stoppers to Create Detection Zone amp Apply Collagen I Overlay Incubate and Allow Cells to Invade into Detection Zone Analyze Cells in Detection Zone Microplate Reader Analysis Detection Mask Attached Image Analysis No Mask Required Seed amp Adhere Cells onto Oris Collagen I Coated Plate Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 RM0039 01 Madis
15. l Free 866 296 4455 RM0039 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 This procedure is intended to assist in determining the cell seeding density needed to achieve confluency of your cell line when using the Oris Collagen I Cell Invasion Assay 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 cm2 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 2 Prepare a log phase culture of the cell line to be tested Collect cells and determine the total number of cells present 3 Pellet cells by centrifugation Prepare three serial dilutions at final concentrations of 1 0 x 106 0 5 x 106 and 0 25 x 106 cells mL 4 Dispense 100 L of cell suspension per well into the 96 well plate to result in the following plate layout 5 Incubate the plate in a humidified chamber 37 C 5 CO2 for 1 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 t
16. ly 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 4 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 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 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 Align the holes in the attachment lugs with the bosses on the bottom of the plate 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
17. microplate reader analysis HT 1080 Cells in OrisTM Collagen I Cell Invasion Assay 500 m Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 RM0039 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 8 VIII ORDERING INFORMATION For a complete list of assays visit Platypus Technologies at www platypustech com order_main html For technical assistance contact Technical Support at 866 296 4455 or techsupport platypustech com IX TERMS amp CONDITIONS Certain uses of these products may be covered by U S Pat No 6 284 197 No 7018838 No 10 597 118 No 11 342 413 and No 60 836 109 licensed to 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
18. on WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 3 II ORISTM PLATE DIMENSIONS Diameter of Well 6 5 mm Diameter of Stopper Space Detection Zone 2 mm Suggested Media Volume per Well populated with Stoppers 100 L Effective Area of Outer Annular Region seeding region per Well 30 03 mm2 Effective Area of Central Detection Zone per Well 3 14 mm2 Plate Height 14 9 mm Plate Height with Lid with OrisTM Cell Seeding Stoppers 23 9 mm Offset of Wells A 1 location X 14 4 mm Offset of Wells A 1 location Y 11 2 mm Distance between Wells 9 mm on center Well Depth 12 2 mm Thickness of Well Bottom 0 25 mm Well Coating Material Collagen I rat tail Storage Conditions Refrigerate 4 C Important Read Instructions Before Performing any OrisTM Assay III MATERIALS PROVIDED Product No CIA101CC Component Quantity Storage Oris Collagen I Coated 96 well Plate with Oris Cell Seeding Stoppers 1 Refrigerate 4 C Oris Detection Mask 1 Room Temperature Oris Stopper Tool 1 Room Temperature Oris Collagen I Stock Reagent 4 mL Refrigerate 4 C Oris 10X PBS Buffer 1 mL Refrigerate 4 C Room Temperature Product No CIA200CC Component Quantity Storage Oris Collagen I Coated 96 well Plates with Oris Cell Seeding Stoppers 2 Refrigerate 4 C Oris Detection Mask 1 Room Temperature Oris St
19. opper Tool 1 Room Temperature Oris Collagen I Stock Reagent 2 x 4 mL Refrigerate 4 C Oris 10X PBS Buffer 2 x 1 mL Refrigerate 4 C Room Temperature Oris Collagen I Stock Reagent must be stored at 4 C for use within 6 months of receipt Do not freeze Oris is a trademark of Platypus Technologies LLC Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 RM0039 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 4 IV MATERIALS REQUIRED Biological Cells 7 5 Sodium Bicarbonate Complete Cell Culture Growth Medium containing serum Sterile PBS containing both Ca and Mg Hanks Balanced Salt Solution HBSS Serum Free Cell Culture Medium 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 e g Calcein AM required if performing staining V PRECAUTIONS AND RECOMMENDATIONS For Research Use Only Not for use in diagnostic procedures Handling and Use of the Oris Collagen I Stock Reagent Please note that it is crucial that the Collagen I Overlay concentration be optimized for cell line and experimental conditions since different cell lines and different experimental conditions can result in varied amounts of cell invasion
20. r MUST be set to read from the bottom of the plate To set up reference controls refer to Section V Precautions and Recommendations Sample Data Obtained via Microscopy and Microplate Reader are shown in Figure 5 Wells were seeded with 30 000 HT 1080 cells i e 100 L of 3 0x105 cells mL and the plate was incubated for 1 hour The stoppers were removed from the wells the wells were rinsed with serum free media and the OrisTM Collagen I Overlay without serum final concentration of 3 mg mL was overlayed on the cell monolayer sixteen 16 wells were left stoppered to represent t 0 reference After polymerization was permitted for 1 hour complete media containing 10 FBS was added on top of the Collagen I Overlay The plate was then incubated in a humidified chamber for 48 hours to permit cell invasion Cells were labeled with Calcein AM and images were captured using a Zeiss Axiovert microscope 5X magnification Fluorescence in the detection zone was quantified by using a microplate reader The images below captured without a detection mask in place illustrate representative data from pre invasion t 0 hrs image 1a and post invasion t 48 hrs image 1b wells The graph depicts the average Relative Fluorescent Units RFU s in the detection zones for each condition each column represents the mean SD of at least 16 wells 1a 1b Pre Invasion Post Invasion 48 hrs Figure 5 Cell invasion data obtained via microscopy and
21. rmined optimal in Appendix I and allow cells to adhere At the end of the assay remove stoppers from the reference wells treat with the Oris Collagen I Overlay incubate to allow for gel formation and proceed with staining analysis of the entire plate Experimental Conditions Please note that cell movement along the X Y axes will likely occur in addition to invasion in the Z axis The degree of X Y movement will vary for different cell lines Recommendations for 10X PBS Buffer 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 RM0039 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 5 VI COLLAGEN I CELL INVASION ASSAY 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 0 5 fetal bovine serum may be used if needed 2 Remove the Oris Collagen I Coated Plate from refrigeration and place on lab bench for 1 hour to allow it to equilibrate to room temperature 3 Visually inspect the underside of the populated 96 well plate to ensure that the Oris Cell Seeding Stoppers are firm
22. t the Collagen I Overlay is not dislodged from the bottom sides of the well The following is an example Fluorescent Staining Protocol to label fixed cells with TRITC phalloidin F actin and DAPI nuclei a To fix one fully seeded 96 well plate prepare 10 mL of fixative solution e g 0 25 glutaraldehyde solution in PBS prepared from 8 glutaraldehyde solution Electron Microscopy Sciences b Remove media and rinse wells with 100 L of PBS c Remove PBS and add 100 L of a fixative solution 0 25 glutaraldehyde solution in PBS to each well and incubate at room temperature for 15 minutes d Remove fixative solution and rinse wells with 100 L of PBS e Remove PBS and replace with 100 L of a 1 50 1 100 dilution of TRITC phalloidin Sigma prepared as 10 M stock in methanol in PBS containing 0 1 Triton X 100 f Incubate plate at room temperature for 45 minutes protect from light g Remove the TRITC phalloidin and add 100 L of a 1 4000 dilution of DAPI ThermoScientific in PBS h Incubate plate at room temperature for 2 10 minutes protect from light i Remove DAPI stain and wash wells 2x for 5 minutes each with 200 L of PBS j Replace final wash with 200 L of fresh PBS NOTE This protocol outlines double labeling of cells with a cytoskeletal and a nuclear stain The protocol can be simplified if only one stain is used Substitutions or additional cytostaining or immunostaining may be per
23. th a fluorescent stain after sufficient invasion has occurred Refer to Section VII and Appendices II amp III 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 VII DATA ACQUISITION The readout of the Oris Collagen I Cell Invasion Assay can be conducted at any time allowing the user to perform a kinetic assay or an endpoint assay The Oris Collagen I Cell Invasion Assay 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 instrument Microscope Analysis Cell counting or image analysis software such as NIH ImageJ freeware can be used Note Microscopic observations are possible using phase contrast or fluorescence microscopy No need to attach the Oris Detection Mask to the Oris microplate To set up reference controls refer to Section V Precautions and Recommendations Microplate Reader Analysis Attach the Oris Detection Mask to the bottom of the Oris microplate see Step 4 of Protocol 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 reade
24. uorescence 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 invasion and post invasion wells Refer to the instrument manual for your microplate reader for further guidance on instrument settings At this point you have successfully labeled your live cells Column 1 2 3 Cells well 100 000 50 000 25 000 Number of wells 8 8 8 Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 RM0039 01 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 10 APPENDIX III Fluorescent Labeling Fixed Cell Options This procedure is intended to assist in obtaining data from the Oris Collagen I Cell Invasion Assay using various fluorescent labels The Oris Collagen I Cell Invasion Assay has been designed to work with all types of fluorescent stains and staining techniques The precise method for treating cells with fluorescent stains varies according to the nature of the individual reagent It is important to use 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 NOTE Use caution when adding removing solutions so tha
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