OrisTM Collagen I Cell Invasion Assay
Contents
1. atan angle and viewed under indirect light to reveal the bullseye pattern at the bottom of each well Figure 2 Stoppers that are 4 Apply the Oris Detection Mask to the bottom of the 96 well plate if microplate A Partially Sealed reader data is being collected The Detection Mask is not necessary if collecting B Unsealed imaging 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 see Figure 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 O 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 N assays using fixed and stained cells it is often most convenient to apply the mask just before reading assay results EE T an o aa ee 2 ee eceeee r 2 86 6 a g j r E E EE r2. 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 performed 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 www amsbio com amsbio info amsbio com
3. 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 PURCHASER PLATYPUS shall not be liable for the lack o
4. E 8 6E E Chamfer Attachment Lugs Figure 3 Features of Detection Mask 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 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 V Precautions and Recommendations 7 Pipette 100 uL of suspended cells into each test well through one of the side ports of the Oris Cell Seeding Stopper cor NOTE For best results add or extract media by placing the pipette tip along the wall of the well see Figure 4 Care should k be taken not to disturb the Oris Cell Seeding Stopper or the Collagen coating when introducing the pipette tip into the well A slender elongated tip or a gel loading tip may be useful www amsbio com amsbio info amsbio com IMPORTANT Lightly tap the plate on your work surface to evenly distribute well contents ex
5. Treated 1 pack CMACC1 101 Collagen Coated g o pack CMACC5 101 Oris Cell Seeding Stoppers Oris Cell Migration Assays pre populated Fibronectin Coated IPASE MACN EINT 5 pack CMAFN5 101 1 pack CMATR1 101 meee 5 pack CMATR5 101 Universal 1 pack CMAU101 Oris Cell Migration Tissue Culture Treated 5 pack CMAU505 Oris Cell Seeding Stoppers Assembly Kits not pre populated FLEX Tissue Culture Treated 4 pack CMAUFL4 i aaa Collagen 1 pack CIA101CC Oris Cell Seeding Stoppers Oris Cell Invasion 2 pack CIA200CC pre populated Assays 1 pack CIA101DE Oris Cell Seeding Stoppers 2 pack CIA2Z00DE not pre populated TERMS amp CONDITIONS Certain uses of these products may be covered by U S Pat No 7 018 838 No 10 597 118 No 11 342 413 No 11 890 740 and No 12 195 007 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 limita
6. 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 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 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 www amsbio com amsbio info amsbio com APPENDIX Ill Fluorescent Labeling Fixed Cell Options This procedure is intended to assist in obtaining data from the Oris Collagen Cell Invasion Assay using various fluorescent labels The Oris Collagen 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
7. PLAT YRU2 Bringing Science to the Surface Oris Collagen Cell Invasion Assay Product No CIA101CC amp CIA200CC 96 well 3 D Assay for Investigating Cell Invasion of Adherent Cell Lines on Collagen PROTOCOL amp INSTRUCTIONS INTRODUCTION ORIS PLATE DIMENSIONS MATERIALS PROVIDED MATERIALS REQUIRED PRECAUTIONS AND RECOMMENDATIONS COLLAGEN I CELL INVASION ASSAY PROTOCOL DATA ACQUISITION ORDERING INFORMATION TERMS amp CONDITIONS APPENDIX I Determining Optimal Cell Seeding Concentration APPENDIX II Fluorescent Labeling Live Cell Options APPENDIX III Fluorescent Labeling Fixed Cell Options SP0036 01 www amsbio com amsbio info amsbio com ORIS COLLAGEN I CELL INVASION ASSAY INTRODUCTION The Oris Collagen 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 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 Cel
8. ate 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 overlay 15 Add 100 uL of complete media containing serum on top of the 3 D Collagen Overlay Optional Invasion inhibitors or stimulants may be added to the media www amsbio com amsbio info amsbio com IMPORTANT Use caution when adding media so as not to dislodge the Collagen 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 with 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 tiz N NOTE Oris Cell Seeding Stoppers are for single use only Platypus cannot guarantee the integrity of the stopper material a after a second sterilization procedure VII DATA ACQUISITION The readout of the Oris Collagen Cell Invasion Assay can be conducted at any time allowing the user to perform a kinetic assay or an endpoint assay The Oris Collagen Cell Invasion Assay is designed
9. d 0 25 x 10 cells mL 4 Dispense 100 uL of cell suspension per well into the 96 well plate to result in the following plate layout Column 1 O o Y 2 O 3 Cells well oo 000 50 on 25 000 Number of wells 8 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 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 __ 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 Cell Invasion Assay via colorimetric analysis or microscopy you have successfully determined the optimal cell seeding concentration to be used i
10. ells at different concentrations Seed test wells at a density determined optimal in Appendix 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 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 Overlay incubate to allow for gel formation and proceed with staining analysis of the entire plate To establish t O 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 l allow cells to adhere for 1 18 hours remove stoppers treat with the Oris Collagen 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 determined optimal in Appendix and allow cells to adhere At the end of the assay remove stoppers from the reference wells treat with the Oris Collagen Overlay incubate to allow for gel formation and proceed with staining analysis of the entire plate Experimental C
11. epresent t 0 reference After polymerization was permitted for 1 hour complete media containing 10 FBS was added on top of the Collagen 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 HT 1080 Cells in Oris Collagen Cell Invasion Assay 1a 1b T kea w gt Ww pm Uu om Pre Invasion Post Invasion 48 hrs Pre invasion Invasion 48 hrs Figure 5 Cell invasion data obtained via microscopy and microplate reader analysis www amsbio com amsbio info amsbio com VIII IX ORDERING INFORMATION 1 pack PROCMA1 aye Sap Tissue Culture Treated 5 pack PROCMAS Cell Migration Assays GalsdanlGesied 1 pack PROCMACC1 g 5 pack PROCMACCS Oris Pro 1 pack PROCIACC1 l Cell Invasion Assays woteden lCoatea 2 pack PROCIACC2 Biocompatible Gel Biocompatible Gel 1 pack CMA1 101 5 pack CMA5 101 Tissue Culture
12. f 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 www amsbio com amsbio info amsbio com 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 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 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 2 Prepare alog 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 10 0 5 x 10 an
13. l 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 Cell Invasion Assay kit has been uniquely designed to detect cellular migration and invasion in vitro within a 3 dimensional extracellular matrix comprised of Collagen rat tail The Oris Collagen 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 Cell Invasion Assay offers the following features amp benefits e Membrane free Invasion perform studies without e Versatile analyze data using multiple probes in manipulating transmembrane inserts no membrane a single well by using a microscope digital to restrict the ability to image cells imager or fluorescence microplate reader e Reproducible Results obtain low well to well CV s e Flexible perform real time or endpoint cell due to the unique assay design invasion assays without the use of special e Preserve Cell Morphology realize a more native instrumentation 3 D environment Seed amp Adhere Remove Stoppers Incubate and Allow Analyze Cells in Cells onto Oris to Create Detection Cells to Invade into Detection Zone Collagen Zone am
14. lution 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 e Oris 10X PBS sterile e 7 5 sodium bicarbonate sterile e Deionized water sterile e 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 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 Overlay solution The following example protocol provides volumes for a 3 0 mg mL Collagen Overlay solution 1 4625 mL deionized water 0 5 mL Oris 10X PBS buffer 0 0375 mL 7 5 sodium bicarbonate 3 mL _Oris Collagen I Stock Reagent 5mg mL 5 0 mL total volume i fa A NOTE Supplements such as growth factors may be mixed with the 3 D Collagen Overlay 13 Remove media from wells and add 40 uL of the Oris Collagen I Overlay to each well IMPORTANT Place pl
15. n Step 6 of the Oris Collagen Cell Invasion Assay Protocol APPENDIX Il Fluorescent Labeling Live Cell Options This procedure is intended to assist in obtaining data from the Oris Collagen Cell Invasion Assay using various fluorescent labels The Oris Collagen 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 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 al C Y NOTE Use caution when adding removing solutions so that the Collagen Overlay is not dislodged from the bottom sides j 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 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 b Carefully remove culture medium from wells c Wash wells with 100 uL of PBS containing both Ca and Mg d Add 100 uL
16. ock Reagent Refrigerate 4 C Refrigerate 4 C Oris 10X PBS Buffer Room Temperature Oris Collagen Stock Reagent must be stored at 4 C for use within 6 months of receipt Do not freeze www amsbio com amsbio info amsbio com 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 e 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 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 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 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 c
17. onditions 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 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 www amsbio com amsbio info amsbio com 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 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 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 ha i O NOTE The sealing of the stoppers can be most easily observed if the plate is tipped
18. p Apply Detection Zone Microplate Reader Coated Plate Collagen Overlay Analysis Detection Mask Attached Image Analysis No Mask Figure 1 Schematic of Oris Collagen Cell Invasion Assay Required www amsbio com amsbio info amsbio com ll ORIS PLATE DIMENSIONS Diameter of Stopper Space Detection Zone 2mm Suggested Media Volume per Well populated with Stoppers 100 uL 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 2mm 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 Oris Assay lll MATERIALS PROVIDED Product No ClA101CC Component Quantity Storage Oris Collagen Coated 96 well Plate with 7 Oris Cell Seeding Stoppers Refrigerate 4 C Oris Detection Mask Room Temperature Oris Stopper Tool Room Temperature Oris Collagen Stock Reagent Refrigerate 4 C Oris 10X PBS Buffer 1 mL Regaal oy Room Temperature Product No CIA200CC Oris Collagen Coated 96 well Plates l o with Oris Cell Seeding Stoppers REETA Oris Detection Mask Room Temperature 1 Room Temperature Oris Stopper Tool Oris Collagen St
19. tions 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 are in lieu of all other liability or obligations of
20. 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 e Cell counting or image analysis software such as NIH ImageJ freeware can be used e Note 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 V Precautions and Recommendations Microplate Reader Analysis e Attach the Oris Detection Mask to the bottom of the Oris microplate see Step 4 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 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 e Wells were seeded with 30 000 HT 1080 cells i e 100 uL of 3 0x10 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 Oris Collagen Overlay without serum final concentration of 3 mg mL was overlayed on the cell monolayer sixteen 16 wells were left stoppered to r
21. treme 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 con NOTE It may be necessary to wash the Oris Stopper Tool with 70 y 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 stoppers Figure 5 Removal of Stoppers Panels A B and C Do not use the Oris Stopper Tool as a lever to pry the stoppers from Position the Tines of the Stopper Tool between the Stopper Tips D Lift Vertically the well see Figure 5E as doing so may cause displacement of and E Do NOT Pry Stoppers seeded cells and may distort the detection zone area 11 Remove media with a pipette and gently wash wells with 100 uL 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 So
22. variability of results and reduce correlation between fluorescence signal and cell migration Please consult the manufacturer of your fluorescent stain for specific considerations wie AP NOTE Use caution when adding removing solutions so that the Collagen 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 uL of PBS c Remove PBS and add 100 uL 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 uL of PBS e 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 f Incubate plate at room temperature for 45 minutes protect from light g Remove the TRITC phalloidin and add 100 uL 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 uL of PBS j Replace final wash with 200 uL of fresh PBS
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