OrisTM Cell Migration Assay
Contents
1. amsbio info amsbio com CELL MIGRATION ASSAY PROTOCOL The following steps should be performed in a biological hood using aseptic technique to prevent contamination ja 4 _ ce gt on Remove the Oris Cell Migration Assay Plate 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 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 in 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 is2. Figure 6B Endpoint Detection of HT 1080 Cell Migration into Migration Zone Pre Migration Post Migration t 0 hrs t 24 hrs Pre Migration Post Migration N 8 Condition Figure 6 Cell migration data obtained using Wright Giemsa colorimetric stain www amsbio com amsbio info amsbio com 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 ll and the equipment user manual for your particular instrument e The microplate reader MUST be set to read from the bottom of the plate e Sample data using a fluorescent stain and microplate reader analysis are shown in Figure 7 Wells populated with Oris Cell Seeding Stoppers were seeded with 50 000 HT 1080 cells i e 100 ul of 5x10 cells mL and incubated overnight 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 detection zones were measured using a plate reader The images below Figure 7A captured without a detection mask in place illustrate representative data from pre migration t 0 hrs and post migration t 21
3. center of each well i e the detection zone into which the seeded cells may then migrate The Oris Detection Mask is applied to the plate bottom and restricts visualization to the detection zones allowing only cells that have migrated to be detected see Figure 1 The Oris Cell Migration Assay 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 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 NMuMG 3T3 Swiss albino HCEC HUVEC and MCF10A cell lines Using the Oris Cell Migration Assay offers the following features amp benefits e Membrane free Migration perform studies without e Versatile analyze data using multiple probes in a manipulating transmembrane inserts single well by using a microscope digital imager or e Reproducible Results obtain well to well CV s fluorescence microplate reader lt 12 due to the unique design e Flexible perform kinetic or endpoint cell migration e Preserves Cell Morphology monitor changes in assays without the use of special instrumentation cell structure in real time Seed amp Adhere Remove Allow Cells to Analyze Cells in Detection Cells onto Stoppers to Migrate into Zone Microplate Reader Oris Plate Create Detection Detection Zone Analy
4. hrs wells The graph depicts a real time analysis of cell migration that was prepared by transposing the fluorescent signal into cell numbers Figure 7B Endpoint Detection of HT 10 _ Kinetic Detection of HT 1080 a a Fo Cell Migration into Migration Zone Average Pre Migration Post Migration t O hrs t 21 hrs 10 15 20 Time hrs n 9 wells time point Figure 7 Cell migration data obtained using CellTracker Green fluorescent stain www amsbio com amsbio info amsbio com VII ORDERING INFORMATION Product Name Product Number Product Description Oris Cell Migration Assays CMA1 101 1 pack Oris 96 well Plate with Oris Cell Seeding Stoppers 1 CMA5 101 5 pack Oris 96 well Plates with Oris Cell Seeding Stoppers 5 Oris Detection Mask 1 Oris Cell Migration Assays Collagen Coated Oris Cell Migration Assays Fibronectin Coated Oris Cell Migration Assays TriCoated Oris Universal Cell Migration Assembly Kits Oris Cell Migration Assembly Kits Collagen Coated Oris Cell Migration Assembly Kit FLEX Oris Cell Invasion amp Detection Assays Oris Detection Mask 1 amp Oris Stopper Tool 1 CMACC1 101 1 pack Oris 96 well Collagen Coated Plate with Oris Cell Seeding Stoppers 1 Oris Detection Mask 1 amp Oris Stopper Tool 1 CMAFN1 101 1 pack Oris 96 well
5. Fibronectin Coated Plate with Oris Cell Seeding Stoppers 1 Oris Detection Mask 1 amp Oris Stopper Tool 1 CMATR1 101 1 pack Oris TriCoated 96 well Plate with Oris Cell Seeding Stoppers 1 32 wells Tissue Culture Treated 32 wells Collagen coated 32 wells Fibronectin coated Oris Detection Mask 1 amp Oris Stopper Tool 1 CMAU101 1 pack Oris compatible 96 well Plate 1 Oris Cell Seeding Stoppers 96 Oris Detection Mask 1 amp Oris Stopper Tool 1 CMAUCC1 1 pack Oris Collagen Coated 96 well Plate 1 Oris Cell Seeding Stoppers 96 Oris Detection Mask 1 amp Oris Stopper Tool 1 CMAUFL4 1 kit Oris M compatible 96 well Plates 4 Oris Cell Seeding Stoppers 4 packs of 24 Oris Detection Mask 1 amp Oris Stopper Tool 1 CIA101DE 1 pack Oris 96 well plate 1 Oris BME Stock Reagent 5 mL Oris Cell Seeding Stoppers 96 Oris Detection Mask 1 amp Oris Stopper Tool 1 Calcein AM Reagent 20 uL www amsbio com Oris Stopper Tool 1 CMACC5 101 5 pack Oris 96 well Collagen I Coated Plates with Oris Cell Seeding Stoppers 5 Oris Detection Mask 1 amp Oris Stopper Tool 1 CMAFN5 101 5 pack Oris 96 well Fibronectin Coated Plates with Oris Cell Seeding Stoppers 5 Oris Detection Mask 1 amp Oris Stopper Tool 1 CMATR5 101 5 pack Oris TriCoated 96 well Plates with Oris Cell Seeding Stoppers 5 32 wells T
6. 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 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 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 Chamfer Attachment Lugs Figure 3 Features of Detection Mask Figure 4 Media is Added with Single or Multi Channel Pipette NOTE For
7. amsbio Oris Cell Migration Assay Product No CMA1 101 amp CMA5 101 96 well 2 D Assay for Investigating Cell Migration of Adherent Cell Lines PROTOCOL amp INSTRUCTIONS Table of Contents INTRODUCTION ORIS PLATE DIMENSIONS MATERIALS PROVIDED MATERIALS REQUIRED CELL MIGRATION ASSAY PROTOCOL DATA ACQUISITION ORDERING INFORMATION TERMS amp CONDITIONS APPENDIX I Determining Optimal Cell Seeding Concentration APPENDIX II Determining Optimal Fluorescence Plate Reader Settings 9 RM0007 02 AMSBIO www amsbio com info amsbio com Ar UK amp Rest of the World Wi North America Germany Switzerland ESS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 Oris CELL MIGRATION ASSAY I 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
8. assay The Oris Cell Migration Assay is designed to be used with any commercially available stain or labeling technigue The readout can be performed by using a microscope a microplate reader or a High Content Screening or High Content Imaging Analysis platform Microscope Analysis e Cell counting or image capture analysis software such as NIH ImageJ freeware can be used e Note Microscopy observations are possible using phase contrast or bright field microscopy e No need to attach the Oris Detection Mask to the Oris plate e Sample data using a colorimetric stain is shown in Figure 6 Wells populated with Oris Cell Seeding Stoppers were seeded with 50 000 HT 1080 cells i e 100 ul of 5x10 cells mL and incubated overnight 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 chamber 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 detection mask in place illustrate representative data from pre migration t 0 hrs and post migration t 24 hrs wells Figure 6A The graph depicts the average pixel number in the detection zones for each condition
9. 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 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 Remove plate from incubator www amsbio com amsbio info amsbio com V CELL MIGRATION ASSAY PROTOCOL continued 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 wiz lt NOTE It may be necessary to wash the Oris Stopper Tool with 70 ethanol as the Stopper Tool is not sterile e Secure the 96 well plate by holding it firmly against the deck of your work space Slide the tines of the Oris Stopper Tool under the backbone of the stopper strip keeping the underside of the Oris Stopper Tool flush with the top surface of the plate e Lift the Oris Stopper Tool vertically to gently remove the stoppers NOTE DO NOT use
10. issue Culture Treated 32 wells Collagen coated 32 wells Fibronectin coated Oris Detection Mask 1 amp Oris Stopper Tool 1 CMAU505 5 pack Oris M compatible 96 well Plates 5 Oris Cell Seeding Stoppers 5 x 96 Oris Detection Mask 1 amp Oris Stopper Tool 1 CMAUCC5 5 pack Oris Collagen I Coated 96 well Plates 5 Oris Cell Seeding Stoppers 5 x 96 Oris Detection Mask 1 amp Oris Stopper Tool 1 sufficient materials for 96 tests CIA200DE 2 pack Oris 96 well plates 2 Oris BME Stock Reagent 2 x 5 mL Oris Cell Seeding Stoppers 2 x 96 Oris Detection Mask 2 amp Oris Stopper Tool 2 Calcein AM Reagent 2 x 20 uL 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 Cell Migration 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 a log phase culture of the cell line
11. otocol and microplate reader settings for analysis of cell migration using a fluorescence microplate reader AMSBIO www amsbio com info amsbio com ire UK amp Rest of the World HEE North America Germany ra Switzerland aS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 441 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218
12. sis Detection Mask Zone Attached Image Analysis No Mask Required Figure 1 Schematic of Oris Cell Migration Assay ll ORIS PLATE DIMENSIONS Plate Height 14 9 mm Plate Height with Lid with Oris 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 Thickness of Well Bottom 0 25 mm Storage Conditions Refrigerate 4 C Important Read Instructions Before Performing any Oris Assay www amsbio com amsbio info amsbio com ll MATERIALS PROVIDED Product No CMA1 101 Product No CMA5 101 Oris 96 well Tissue Culture Treated black clear Oris M 96 well Tissue Culture Treated black clear bottom Plate with Oris Cell Seeding Stoppers 1 bottom 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 Ca and Mg Complete Cell Culture Growth Medium containing serum Sterile Pipette Tips Pipette or Multi Channel Pipette Trypsin or Cell Scraper Inverted Microscope optional Fluorescence Microplate Reader optional Cell Culture Labeling Medium phenol red free serum free media Cell Labeling Fluorescent Agent eg CellTracker Green Calcein AM required if performing assay readout via microplate reader www amsbio com
13. 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 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 Figure 5 Removal of Stoppers Panels A B and C Position h the Tines of the Stopper Tool between the Stopper permit cell migration Cells may be examined microscopically Tips D Lift Vertically and E Do NOT Pry throughout the incubation period to monitor progression of Stoppers migration Migration time will vary depending upon cell type and experimental design 14 Incubate plate in a humidified chamber 37 C 5 CO2 to 15 If performing an endpoint analysis of cell migration stain cells with a fluorescent stain after sufficient migration has occured Refer to Section VI and Appendix ll for further information on data acquisition and fluorescence staining technique Y NOTE Oris Cell Seeding Stoppers are for single use only We cannot guarantee the integrity of the s topper material after a second sterilization procedure www amsbio com amsbio info amsbio com 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
14. 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 and 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 2 3 Cells well 100 000 50 000 25 000 Number of wells 8 _ 8 8 5 Incubate the plate in a humidified chamber 37 C 5 CO2 for 4 18 hours cell line dependent with cell seeding stoppers in place to allow the cells to firmly attach to the well surface 6 Following cell attachment remove the Oris Cell Seeding Stoppers from each well see Figure 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 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 yields a confluent monolayer at the perimeter of the detection zone At this point if you plan
15. to obtain the results of the Oris Cell Migration Assay via colorimetric or microscopy analysis you have successfully determined the optimal cell seeding concentration to be used in Step 5 of the Cell Migration Assay 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 1 Using the optimal cell seeding concentration determined in Appendix perform a cell migration assay per Section V Cell Migration Assay Protocol using culture conditions expected to result in robust cell migration Be sure to include equal numbers of pre migration reference wells stoppers left in place until staining and post migration test wells stoppers removed after cell attachment period A minimum of 8 wells per condition are recommended 2 Perform the desired fluorescent staining technique The Oris Cell Migration Assay 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 cons
16. ult the manufacturer of your fluorescent stain for specific considerations The following is an example Fluorescent Staining Protocol for using Calcein AM a To stain one fully seeded 96 well plate combine 5 ul of Calcein AM 1 mg mL in dry DMSO with 10 mL of phenol red free and serum free media or 1x PBS containing both Ca and Mg Protect diluted Calcein AM solution from light until ready to use in step d Carefully remove culture medium from wells Wash wells with 100 ul of PBS containing both Ca and Mg Add 100 ul of diluted Calcein AM solution to each well Incubate plate at 37 C for 30 60 minutes Attach mask and read promptly with microplate reader using appropriate filter set and sensitivity gain settings for a Biolek Synergy HT microplate reader use 485 528 nm excitation emission filters sensitivity 55 nm ooou 3 If not already in place apply the Oris Detection Mask to the plate Using the bottom probe of a fluorescence microplate reader obtain the fluorescence reading from each well To achieve the optimal dynamic range adjust the instrument settings e g gain to result in the greatest difference in fluorescence signal between pre migration and post migration wells Refer to the instrument manual for your microplate reader for further guidance on instrument settings You have now successfully determined the optimal cell seeding concentration to be used in Step 5 of the Cell Migration Assay Pr
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