Cell Migration Assay Kit*
Contents
1. and endothelial HCEC and MCF10A cell lines Using the Oris Cell Migration Assay offers the following benefits e Membrane free Migration no transwell inserts to e Versatile analyze data using multiple probes in a manipulate single well using a microscope digital imager or e Reproducible Results the unique design provides fluorescence plate reader well to well CV s lt 12 e Flexible perform kinetic or endpoint cell migration e Preserves Cell Morphology changes in cell assays without the use of special instrumentation structure can be monitored in real time Fl FI Apply Mask Incubate to Allow Remove Incubate to Seeded Cells that W Observe and Add Cell Attachmentin Stoppers Allow Cells to HAVE NOT hoor Migrated Cells to Outer Annular Migrate into Migrated into Cells Using Wells Region of Wells Migration Central Migration Microscope Zone Zone are Blocked or Plate from View Reader Figure 1 Schematic of Oris Cell Migration Assay PRODUCT SPECIFICATIONS Diameter of Well Diameter of Stopper Space Migration Zone Suggested Media Volume per Well populated with Stoppers 100 ul Effective Area of Outer Annular Region seeding region per Well 30 03 mm Effective Area of Central Migration Zone per Well Storage Conditions Refrigerate 4 C MATERIALS PROVIDED e One 1 96 well Plate with Oris Cell Seeding Stoppers e One 1 Oris Stopper Removal Tool e One 1 Oris Migration Mask MATERIALS REQUIRED2. e Biological Cells e Inverted Microscope optional e Cell Culture Medium e Fluorescence Microplate Reader optional e Sterile PBS e Cell Labeling Fluorescent Agent eg CellTracker e Sterile Pipette Tips and Pipette or Multi Channel Green Calcein AM required if performing assay Pipette readout via plate reader a product of Molecular e Trypsin or Cell Scraper Probes Invitrogen PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 2 PLATYPUS V CELL MIGRATION ASSAY PROTOCOL l 10 11 Remove the Oris Cell Migration Assay from 4 C and place on lab bench for 1 hour to allow it to equilibrate to room temperature Visually inspect the bottom 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 sealing is not 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 Figure 2 Partially Sealed A Unsealed B at an angle and viewed under indirect light looking for the bullseye pattern at the and Completely Sealed C Stoppers bottom of each well Aperture Orientation A 1 Corner Apply the Oris Migration Mask to the bottom of the 9
3. cell number for your first serial dilution Cells well 50 000 25 000 12 500 Number of wells 4 Incubate the plate in a humidified chamber 37 C 5 CO2 for 16 hours with cell seeding stoppers in place 5 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 adhered cells e Secure the 96 well plate by holding it firmly against the deck of your work space Slide the tines of the removal tool under the backbone of the stopper strip keeping the underside of the removal tool flush with the top surface of the plate e Lift the removal tool vertically to gently remove the stopper Do not use the removal tool as a lever to pry the stoppers from the well as doing so may cause displacement of the seeded cells 6 Use a microscope to visually inspect the cells and determine the cell seeding concentration that yields a confluent layer NOTE If you plan to obtain the results of the Oris Cell Migration Assay via colorimetric or microscopic analysis you have successfully determined the optimal cell seeding concentration for your cell line Proceed to Step 2 of the Cell Migration Assay Protocol If you plan to obtain the results of the Oris Cell Migration Assay via a fluorescence plate reader proceed with the following steps to optimize your plate reader settings 7 The Oris Cell Migration Assay has been designed to work with all types of fluorescence
4. pack Seeding Stoppers 1 Oris Migration Mask amp 1 Oris Stopper Removal Tool Oris Cell Migration Assay Collagen Coated 5 pack CTM z 1 101M CMACCS5 101 5 Oris Collagen I Coated 96 well plates black clear bottom with Oris 5 pack Cell Seeding Stoppers 1 Oris Migration Mask amp 1 Oris Stopper Removal Tool To place an order visit the Platypus Technologies website at www platypustech com order_main html For technical assistance contact Technical Support at 866 296 4455 or techsupport platypustech com 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 produc
5. stains and staining techniques The precise method for staining cells with fluorescence stains varies according to the nature of the individual stain Please consult the manufacturer of your fluorescence stain for specific considerations First Time Users For a guide to using Calcein AM see below a Aspirate media from wells amp wash wells with PBS or media b Add 100 ul of Calcein AM to each well at an appropriate concentration for a fully seeded 96 well plate combine 5 ul of reconstituted Calcein AM 1mg mL in dry DMSO with 10 mL of serum free media or 1x PBS c Incubate plate at 37 C for 20 minutes d Remove plate from incubator e Aspirate staining solution f Fix cells or to prevent drying add 100 ul of 1x PBS to each well 8 Apply the Oris Migration Mask to the plate 9 Using the bottom probe of a fluorescence plate reader obtain the total output from each well adjust the gain settings to achieve optimal dynamic range To determine optimal dynamic range consider the following factors a The gain setting that permits detection of the lowest concentration of cells b The gain setting that permits discrimination between cell numbers at higher densities NOTE When using a plate reader to analyze the Oris Cell Migration Assay it is important to stain cells using a fluorescence reagent that uniformly stains cells The use of a fluorescence probe that is affected by experimental conditions will increase variability of
6. 6 well plate First Time Users In order to prevent splashing of well contents familiarize yourself with the attachment and removal of the Migration Mask before any liquids are placed in the wells e Orient the chamfered corners of the mask with those of the 96 well plate ensuring that the Al corner of the mask is aligned with the Al 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 A mask is not sterile The mask may be applied at any point during the assay For kinetic assays Chamfer Attachment Lugs 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 Figure 3 Features of Migration Mask to apply the mask just before reading assay results If performing a kinetic analysis of cell migration pre stain 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 see Appendix I for a discussion of this process Pipet
7. Cell Migration Assay Kit 96 well 2 D Assay for Investigating Cell Migration of Adherent Cell Lines PROTOCOL amp Instructions for use Patent Pending PLAT Y POS FEC HNOLOGTES ELE Pa OF NOD ELD RI tS ULE O MADISON WI 53711 TOLL FREE 866 296 4455 PHONE 608 237 1270 FAX 608 237 1271 WWW PLATYPUSTECH COM PLATY PUS Had PA Bringing Science to the Surface Oris CELL MIGRATION ASSAY INTRODUCTION The Oris Cell Migration Assay 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 1 e the migration zone into which the seeded cells may then migrate The Oris Migration Mask is applied to the plate bottom and restricts visualization to the migration zones thus allowing only migrated cells to be detected see Figure 1 The Oris Cell Migration Assay is designed to be used with any commercially available stain or labeling technique and the readout can be performed by microscopic examination or by using a plate reader The Oris Cell Migration Assay system has been designed for use with adherent cell cultures This assay has been successfully used with fibroblast NIH 3T3 fibrosarcoma HT 1080
8. amber for 24 hours to permit cell migration Stoppers were removed from the reference wells and all cells were fixed and treated with Wright Giemsa stain Images were captured using bright field microscopy and then imported to Image J software for analysis using thresholding The images below captured without a migration mask in place illustrate representative data from pre migration t O hrs and post migration t 24 hrs wells The graph depicts the average pixel number in the migration zones for each condition Endpoint Detection of HT 1080 Cell Migration into Migration Zone 250000 200000 T 150000 P QO 100000 50000 Pre Migration Post Migration P a t 0 hrs t 24 hrs Pre Migration Post Migration N 8 Condition Plate Reader Analysis e Setup on individual plate readers varies according to make and model Consult your user manual for proper operation e The plate reader MUST be set to use the bottom probe read e Sample Data using a fluorescent stain is shown below Wells were seeded with 50 000 HT 1080 cells i e 100 ul of 5x10 cells mL and incubated for 4 hours The stoppers were removed from test wells but remained in place in the pre migration reference wells until the time of the assay readout Cells were fluorescently stained with CellTracker Green The seeded plate was incubated in a humidified chamber for 28 hours and at various time points the fluorescence signals in the migration zones w
9. cally and E Do NOT Pry Stoppers PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 3 A NOTE DO NOT use the removal tool as a lever to pry the stoppers from the well as doing so may cause displacement of seeded cells 12 Remove media and gently wash wells with 100u PBS or media to remove any unattached cells 13 Add appropriate amount of fresh culture media to each well 14 Incubate plate in a humidified chamber 37 C 5 CO to permit cell migration Incubation time will vary depending upon cell type and experimental design 15 If performing an endpoint analysis of cell migration apply stain VI DATA ACQUISITION The readout of the Oris Cell Migration Assay can be conducted at any time allowing the user to perform a kinetic assay or an endpoint assay The Oris Cell Migration Assay is designed to be used with any commercially available stain or labeling technique The readout can be performed by microscopic examination or by plate reader Microscopic Analysis e Cell counting or image capture analysis using software such as Image J freeware available from NIH e Sample Data using a colorimetric stain is shown below Wells were seeded with 50 000 HT 1080 cells 1 e 100 ul of 5x10 cells mL and incubated for 4 hours The stoppers were removed from test wells but remained in place in the pre migration reference wells until the time of the assay readout The seeded plate was incubated in a humidified ch
10. ere measured using a plate reader The images below captured without a migration mask in place illustrate representative data from pre migration t 0 hrs and post migration t 21 hrs wells The graph depicts a real time analysis of cell migration that was prepared by transposing the fluorescent signal into cell numbers by employing a standard curve and a 5 Parameter Logistic fit Equation Endpoint Detection of HT 1080 Cell Migration Kinetic Detection of HT1080 ES paeen Fi i PTE ll Migration into Migrati ah 2500 2000 1500 1000 yeh ie te Ea cairn ay 500 PT an a A a o 7 Pre Migration Post Migration o 5 10 15 20 25 30 t 0 hrs t 21 hrs Time hrs n 9 wells time point PLATYPUS TECHNOLOGIES E Bringing Science to the Surface ie Vil ORDERING INFORMATION VIII PLATYPUS Product No Product Description Package Size Oris Cell Migration Assay l pack CMA1 101 1 Oris 96 well plate black clear bottom with Oris Cell Seeding Stoppers 1 pack 1 Oris Migration Mask amp 1 Oris Stopper Removal Tool Oris Cell Migration Assay 5 pack CMAS 101 5 Oris 96 well plates black clear bottom with Oris Cell Seeding Stoppers 5 pack 1 Oris Migration Mask amp 1 Oris Stopper Removal Tool Oris Cell Migration Assay Collagen Coated 1 pack CTM _ j 101M CMACC1 101 1 Oris Collagen I Coated 96 well plate black clear bottom with Oris Cell 1
11. results and reduce correlation between fluorescence signal and cell migration Fluorescence probes that are affected by experimental conditions could be utilized however as counterstains for the study of factors and processes affecting cell migration You have successfully determined the optimal cell seeding concentration for your cell line Proceed to Step 2 of the Cell Migration Assay Protocol PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 6
12. 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 For a comprehensive list of Platypus s Terms amp Conditions access www platypustech com products termsconditions html PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 5 PLATYPUS APPENDIX I Determining Optimal Cell Seeding Concentration This appendix 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 To that end several dilutions of cell suspensions will be investigated NOTE The Oris Migration Mask MUST be removed from the 96 well plate prior to the start of the following steps 1 Collect cells by trypsinization or mechanical scraping and calculate total number of cells Pellet cells by centrifugation and resuspend to a final concentration of 500 000 cells mL in culture media 3 Seed a 100 ul portion of cells at 2 fold serial dilutions in the 96 well plate starting at 50 000 cells well a suggested starting amount as shown below Keep in mind that the cell seeding area of the well with the stopper in place is 0 3 cm and based on the typical seeding density of your cells you can infer the appropriate
13. te 100u1 of suspended cells into each test well through one of the side ports of the Cell Seeding Stopper Figure 4 Media is Added with Single or NOTE For best results add or extract media by placing the pipette tip along the wall of the well Mohe channel FADE tte see Figure 4 Care should be taken not to disturb the Cell Seeding Stopper when introducing the pipette tip into the well 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 CO for 10 to 16 hours cell line dependent to permit cell attachment Remove plate from incubator Designate several reference wells that will represent t O in which the stoppers will remain in place until results are read Using the Oris Stopper Removal Tool remove all other stoppers see Figure 5 e Secure the 96 well plate by holding it firmly against the deck of your work space Figure 5 Removal of Stoppers Slide the tines of the removal tool under the backbone of the stopper strip Panels A B and C Position the Tines of the keeping the underside of the removal tool flush with the top surface of the plate Removal Tool between the Stopper Tips D e Lift the removal tool vertically to gently remove the stopper Lift Verti
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