Universal Cell Migration Assembly Kit Protocol & Instructions
Contents
1. Condition Figure 7 Cell Migration Data using a Colorimetric Stain 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 Sample Data using a fluorescent stain is shown in Figure 8 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 detection zones were measured using a plate reader The images below captured without a detection 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 Kinetic Detection of HT 1080 enepant Detection of HT 1 080 Cell uae dapand oy SE ER EEE Cell Migration into Detection Zone Average Pre Migration Post Migration t 0 hrs t 21 hrs 10 15 20 Time hrs n 9 wells time point Figure 8 Cell Migration2. Orientation A 1 Corner First Time Users In order to prevent splashing of well contents familiarize yourself with the attachment and removal of the Detection Mask before any ia r ii CE liquids are placed in the wells p s ro oo t amp u amp amp e Orient the chamfered corners of the mask with those of the 96 well plate z z z ensuring that the A1 corner of the mask see Figure 4 is aligned with the i senene A1 well of the plate TTTTT e Align the holes in the attachment lugs with the bosses on the bottom of the eeceoes 96 well plate e Gently press the mask until it is flush with the bottom of the 96 well plate AN Chamfer Attachment Lugs 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 Figure 4 Features of Detection Mask 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 PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 4 Using the Oris Stopper Tool remove all other stoppers see PLAT YPUS If performing a kinetic analysis of cell migration pre stain cells to be seeded with a fluorescent stain now Collect cells and prepare a suspension that is 10 fold
3. 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 Remove staining solution Do not aspirate using an in house vacuum f Fix cells or to prevent drying add 100 ul of 1x PBS to each well 9 Apply the Oris Detection Mask to the plate 10 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 Universal Cell Migration Assembly Kit 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 results and reduce correlation between fluorescence signal and cell migration Fluorescence probes that are affected by experimental conditions could be used 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 6 of the Oris Universal Cell Migration Assembly Kit Proto
4. CONDITIONS Certain uses of these products may be covered by U S Pat No 6 284 197 No 7018838 No 10 597 118 No 11 842 413 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 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 confor
5. Data using a Fluorescent Stain Oris is a trademark of Platypus Technologies LLC CellTracker Green is a trademark of Invitrogen Corporation Transwell is a registered trademark of Corning Inc PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 6 VII VIII ORDERING INFORMATION Product No Product Description E Oris Universal Cell Migration Assembly Kit 1 pack Oris compatible 96 well plate black clear bottom 1 CMAU101 Oris Cell Seeding Stoppers 96 1 pack Oris Detection Mask 1 Oris Stopper Tool 1 Oris Universal Cell Migration Assembly Kit 5 pack Oris compatible 96 well plates black clear bottom 5 CMAU505 Oris Cell Seeding Stoppers 5 x 96 5 pack Oris Detection Mask 1 Oris Stopper Tool 1 Oris Cell Migration Assembly Kit Collagen Coated 1 pack Oris Collagen Coated 96 well plate black clear bottom 1 CMAUCC1 Oris Cell Seeding Stoppers 96 Oris Detection Mask 1 Oris Stopper Tool 1 Oris Cell Migration Assembly Kit Collagen Coated 5 pack Oris Collagen Coated 96 well plates black clear bottom 5 CMAUCC5 Oris Cell Seeding Stoppers 5 x 96 Oris Detection Mask 1 Oris Stopper Tool 1 To place an order visit the Platyous 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
6. E IAI A IS Universal Cell Migration Assembly Kit Product No CMAU101 amp CMAU505 96 well Assay for Investigating Cell Migration Cell Invasion and 2 D Closure of Adherent Cell Lines Protocol amp Instructions Patent Pending e PLATYPUS FECHNOLOGTES LEG Pa OPN OD ELD RI G UETEOGO MADISON WI 53711 TOLL FREE 866 296 4455 PHONE 608 237 1270 FAX 608 237 1271 WWW PLATYPUSTECH COM PLATYPUS l l i RM0024 01 Bringing Science to the Surface Oris Universal Cell Migration Assembly Kit I INTRODUCTION The Oris Universal Cell Migration Assembly Kit is a reproducible sensitive and flexible assay that can be used to monitor cell migration cell invasion and 2 D closure The Oris Universal Cell Migration Assembly Kit now gives researchers more control over designing a cell migration assay Each Oris Cell Migration Assembly kit contains the Oris Cell Seeding Stoppers for creating a detection zone at the center of each well in a 96 well plate Since the stoppers are not pre inserted into the wells researchers have two options coat the plate with an extracellular matrix ECM component or use the coated plate provided to design an assay Researchers may also apply 3 dimensional overlays in each well to watch how cells invade and respond to various compounds chemokinesis Each kit is supplied with a 96 well black clear bottom plate an Oris Detection Mask an Oris Stopper Too
7. col PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 8 PLATYPUS
8. ency of your cell line when using the Oris Universal Cell Migration Assembly Kit To that end several dilutions of cell suspensions will be investigated NOTE The Oris Detection Mask MUST be removed from the 96 well plate prior to the start of the following steps Collect cells by trypsinization or by non enzymatic means Such as 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 Populate test wells with Oris Cell Seeding Stoppers see Steps 1 2 of the Protocol Seed 100 ul 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 cell number for your first serial dilution Cells well 50 000 25 000 12 500 nae oc ea Number of wells DO e e 5 Incubate the plate in a humidified chamber 37 C 5 COs for 16 hours with cell seeding stoppers in place 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 adhered cells e Secure the 96 well plate by holding it firmly against the deck of your work space Slide the tines of the stopper tool under the backbone of the stopp
9. er strip keeping the underside of the tool flush with the top surface of the plate e Lift the stopper tool vertically to gently remove the stopper Do not use the tool as a lever to pry the stoppers from the well as doing so may cause displacement of the seeded cells 7 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 Universal Cell Migration Assembly Kit via colorimetric or microscopic analysis you have successfully determined the optimal cell seeding concentration for your cell line Proceed to Step 5 of the Oris Universal Cell Migration Assembly Kit Protocol If you plan to obtain the results of the Oris Universal Cell Migration Assembly Kit via a fluorescence plate reader proceed with the following steps to optimize your plate reader settings 8 The Oris Universal Cell Migration Assembly Kit has been designed to work with all types of fluorescence 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
10. 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 for a discussion of this process Pipette 100 ul of suspended cells into each test well through one of the side ports of the Cell Seeding Stopper NOTE For best results add or extract media by placing the pipette tip along the wall of the Figure 5 Media is Added with well see Figure 5 Care should be taken not to disturb the Cell Seeding Stopper when Single or Multi introducing the pipette tip into the well A gel loading tip may be useful Channel Pipette IMPORTANT Gently 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 COsz for 4 to 16 hours cell line dependent to permit cell attachment Remove plate from incubator Designate several reference wells in which the stoppers will remain in place until results are read t O pre migration controls Figure 6 e Secure the 96 well plate by holding it firmly against the deck of your work space Slide the tines of the stopper tool under the backbone of the stopper strip keeping the underside of the tool flush with the top surface of the plate e Lif
11. l and Oris Cell Seeding Stoppers The Oris Universal Cell Migration Assembly Kit has been designed for use with adherent cell cultures This assay has been successfully used with fibroblast 3T3 Swiss albino fibrosarcoma HT 1080 and endothelial HCEC and MCF10A cell lines The Oris Universal Cell Migration Assembly Kit offers the following benefits e Membrane free Cell Migration or Invasion no e Real time Monitoring changes in cell structure during cumbersome cell culture inserts to manipulate or limit movement can be monitored in real time with a cellular movement there is no Transwell membrane microscope digital imaging system or fluorescence insert to block live images of cell movement plate reader e Creative Assay Design coat any ECM or BME on the e Reproducible Results 2 D closure assays using the plate to create a 2 D or 3 D environment for cell Oris Cell Seeding Stoppers have better reproducibility migration cell invasion or 2 D closure assays than wound healing or scratch assays e Preserve Cell Morphology the Oris assay provides Versatile Detection Modes analyze cells using __ a more native environment since cells do not have to multiple fluorescent probes labels or colorimetric stains penetrate through a polycarbonate membrane cells can in the same well move across a tissue culture treated surface or within a e Flexible Assay Formats design kinetic or endpoint user applied ECM assays witho
12. m 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 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 de
13. nducted at any time allowing the user to perform a kinetic assay or an endpoint assay The Oris Universal Cell Migration Assembly Kit 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 Sample Data using a colorimetric stain is shown in Figure 7 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 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 The graph depicts the average pixel number in the detection zones for each condition Endpoint Detection of HT 1080 Cell Migration into Detection Zone Pre Migration Post Migration t 0 hrs t 24 hrs Pre Migration Post Migration N 8
14. oppers NOTE It is extremely important to ensure that the into Wells and E Fully Inserted Stoppers stoppers are inserted perpendicular to the well bottom and fully engaged with the well bottom Failure to do so will increase the CV of your data set If you require data sets with low CVs potential for lt 12 l it is recommended to use the pre populated Oris Cell Migration Assay kit HCMA1 101 nf i p1 Optional If desired coat the bottom of the wells with an Extracellular Matrix ECM component collagen fibronectin laminin etc and allow the ECM to dry prior to populating the plate with the Oris Cell Seeding Stoppers 2 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 3 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 Figure 3 Stoppers that are A Partially Sealed B Unsealed amp C NOTE the sealing of the stoppers can be most easily observed if the plate is Completely Sealed tipped at an angle and viewed under indirect light looking for the bullseye pattern at the bottom of each well see Figure 3 3 Apply the Oris Detection Mask to the bottom of the 96 well plate Aperture
15. rs 5 x 96 Oris Detection Mask 1 Oris Stopper Tool 1 Inverted Microscope optional Fluorescence Microplate Reader optional Cell Labeling Fluorescent Agent eg CellTracker Green Calcein AM required if performing assay readout via plate reader a product of Molecular Probes Invitrogen Extracellular Matrix ECM or Basement membrane Extract BME for creating a 3 D assay optional pg 3 ORIS UNIVERSAL CELL MIGRATION ASSEMBLY KIT PROTOCOL The following steps should be performed in a biological hood using aseptic technique to prevent contamination 1 Under sterile conditions populate the 96 well plate with Oris Cell Seeding Stoppers Vertically position the tip ends of two 4 stopper strips into one full column of 8 wells at a time Figure 2A e Gently press down on the strip backbone to partially insert the stoppers halfway into the well Figure 2B When both stopper strips have been partially inserted in 1 column ensure that the position of the stoppers is vertical with respect to the well wall making any necessary adjustments Figure 2C e Using the Oris Stopper Tool firmly press down on the strip backbone to fully insert the stoppers into each well Figure 2D and 2E Repeat for all remaining columns Figure 2 Stopper Insertion Process A Placement of Stoppers into Wells B Close up of Stoppers Partially Inserted into Wells C Proper Placement of Stoppers D Pressing of St
16. t the stopper tool vertically to gently remove the stopper NOTE DO NOT use the stopper tool as a lever to pry the stoppers from the well see Figure 6E as doing so may cause displacement of seeded cells and may distort the detection zone area Figure 6 Removal of Stoppers Panels A B and C Position the Tines of the Stopper Tool between the Stopper PBS or media to remove any unattached cells Do not aspirate Tips D Lift Vertically and E Do NOT Pry using an in house vacuum Stoppers Remove media with a pipette and gently wash wells with 100 ul Optional If the plate was originally coated with an ECM in Step 1 an overlay of ECM may be introduced in the wells to facilitate a 3 Dimensional invasion assay Optimization of experimental conditions will be required to establish invasion conditions for a given cell line Add 100 ul of fresh culture media to each well Incubate plate in a humidified chamber 37 C 5 CO2 to permit cell migration Incubation time will vary depending upon cell type and experimental design If performing an endpoint analysis of cell migration apply stain NOTE Oris Cell Seeding Stoppers are for single use only Platypus can not guarantee the integrity of the stopper material after a second sterilization procedure PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 5 Vi DATA ACQUISITION The readout of the Oris Universal Cell Migration Assembly Kit can be co
17. termines in its sole discretion that PURCHASER has altered or misused the goods or has failed to use or store them in accordance with instructions furnished by PLATYPUS PLATYPUS s sole and exclusive liability and PURCHASER s exclusive remedy with respect to goods proved to PLATYPUS s satisfaction applying analytical methods reasonably selected by PLATYPUS to be defective or nonconforming shall be the replacement of such goods free of charge upon the return of such goods in accordance with our instructions although at its discretion PLATYPUS may provide a credit or refund If PLATYPUS manufactures custom goods for PURCHASER based on instructions specifications or other directions provided by PURCHASER PLATYPUS shall not be liable for the lack of sufficiency fitness for purpose or quality of the goods to the extent attributable to such instructions specifications or other directions PLATYPUS shall not be liable for any loss damage or penalty as a result of any delay in or failure to manufacture deliver or otherwise perform hereunder due to any cause beyond PLATYPUS s reasonable control PLATYPUS shall not be liable for injury or damages resulting from the use or misuse of any of its products PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 7 PLATYPUS APPENDIX I Determining Optimal Cell Seeding Concentration This appendix is intended to assist in determining the cell seeding density needed to achieve conflu
18. ut the use of special instrumentation Populate Incubate to Wells with Allow Cell Allow Cellsto that HAVE NOT Migrated Cells Stoppers Add Cells Attachment in Migrate into Migrated into Using to Wells Outer Annular Detection Central Microscope or Region of Wells Zone Detection Zone Plate Reader are Blocked from Figure 1 Schematic of Oris Universal Cell Migration Assembly Kit View Remove Incubate to Seeded Cells Stoppers ll ORIS PLATE DIMENSIONS per well Diameter of Well Diameter of Stopper Space Detection Zone Suggested Media Volume per Well populated with Stoppers Effective Area of Outer Annular Region seeding region per Well Effective Area of Central Detection Zone per Well Important Read Instructions Before Performing any Oris Assay PLATYPUS TECHNOLOGIES Bringing Science to the Surface pg 2 PLATYPUS lll MATERIALS PROVIDED Product No CMAU101 Oris compatible 96 well black clear bottom Plate 1 Oris Cell Seeding Stoppers 96 Oris Detection Mask 1 Oris Stopper Tool 1 IV MATERIALS REQUIRED PLATYPUS Biological Cells Cell Culture Medium Sterile PBS Sterile Pipette Tips and Pipette or Multi Channel Pipette Trypsin or Non Enzymatic Cell Removal Reagent or Scraper PLATYPUS TECHNOLOGIES Bringing Science to the Surface Product No CMAU505 Oris compatible 96 well black clear bottom Plates 5 Oris Cell Seeding Stoppe
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