Oris™ Universal Cell Migration Assembly Kit
Contents
1. PLAT Y RU2 Bringing Science to the Surface Ons Universal Cell Migration Assembly Kit Product No CMAU101 amp CMAUS505 96 well Assay for Investigating Cell Migration and Cell Invasion of Adherent Cell Lines PROTOCOL amp INSTRUCTIONS Table of Contents INTRODUCTION ORIS PLATE DIMENSIONS MATERIALS PROVIDED MATERIALS REQUIRED UNIVERSAL CELL MIGRATION ASSEMBLY PROTOCOL TERMS amp CONDITIONS APPENDIX I Determining Optimal Cell Seeding Concentration APPENDIX II Determining Optimal Fluorescence Microplate Reader Settings 9 Platypus Technologies LLC 5520 Nobel Drive Suite 100 Madison WI 53711 Toll Free 866 3296 4455 Phone 608 237 1270 Fax 608 237 1271 www platypustech com SP0031 04 ORIS UNIVERSAL CELL MIGRATION ASSEMBLY KIT INTRODUCTION The Oris Universal Cell Migration Assembly Kit is a reproducible sensitive and flexible assay that can be used to monitor cell migration or cell invasion 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 can coat the plate with an extracellular matrix ECM component to design their assay Researchers may also apply 3 dimensional overlays in each w2. 1 Oris compatible 96 well black clear bottom Plates 5 Oris Cell Seeding Stoppers 96 Oris Cell Seeding Stoppers 5 x 96 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 Calcium and Magnesium 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 Extracellular Matrix ECM or Basement membrane Extract BME for creating a 2 D coating or a 3 D assay optional Oris is a trademark of Platypus Technologies LLC CellTracker Green is a trademark of Invitrogen Corporation T Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 E SP0031 04 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 3 V UNIVERSAL CELL MIGRATION ASSEMBLY PROTOCOL The following steps should be performed in a biological hood using aseptic technique to prevent contamination 1 Remove the Oris compatible 96 well plate from refrigeration and place on lab bench for 1 hour to allow it to equilibrate to room tem
3. 96 4455 or techsupport platypustech com Vill TERMS amp CONDITIONS Certain uses of these products may be covered by U S Pat No 7 842 499 issued to or patents applied for by 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 it
4. 96 well plate ensuring that the A1 corner of the mask is aligned with the A1 well of the plate see Figure 4 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 eeececees eeeoeeeesck O NOTE It may be necessary to wash the mask with ethanol to remove dust and debris a since the mask is not sterile The mask may be applied at any point during the i assay For kinetic assays it is often most convenient to apply the mask at the 1 beginning of the assay before any liquids are placed in the well For endpoint assays Chamier Attachment Lugs using fixed and stained cells it is often most convenient to apply the mask just before l reading assay results Figure 4 Features of Detection Mask u Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0031 04 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 4 UNIVERSAL CELL MIGRATION ASSEMBLY PROTOCOL continued 5 6 13 14 15 If performing a kinetic analysis of cell migration pre label cells to be seeded 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
5. 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 Perform the desired fluorescent staining technique The Oris Universal Cell Migration Assembly Kit 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 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 w
6. 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 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 Universal Cell Migration Assembly Kit via colorimetric or microscopic analysis you have successfully determined the optimal cell seeding concentration to be used in Step 5 of the Universal Cell Migration Assembly 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 Universal Cell Migration Assembly Kit 1 Using the optimal cell seeding concentration determined in Appendix perform a cell migration assay per Section V Universal Cell Migration Assembly Protocol using culture conditions expected to result in robust cell migration
7. ctive if PLATYPUS determines in its sole discretion that PURCHASER has altered or misused the goods or has failed to use or store them in accordance with instructions furnished by PLATYPUS PLATYPUS s sole and exclusive liability and PURCHASER s exclusive remedy with respect to goods proved to PLATYPUS s satisfaction applying analytical methods reasonably selected by PLATYPUS to be defective or nonconforming shall be the replacement of such goods free of charge upon the return of such goods in accordance with our instructions although at its discretion PLATYPUS may provide a credit or refund If PLATYPUS manufactures custom goods for PURCHASER based on instructions specifications or other directions provided by PURCHASER PLATYPUS shall not be liable for the lack of sufficiency fitness for purpose or quality of the goods to the extent attributable to such instructions specifications or other directions PLATYPUS shall not be liable for any loss 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 ere Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0031 04 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 8 APPENDIX I Determining Optimal Cell Seeding Co
8. e reference wells and all cells were fixed and treated with Wright Giemsa stain Images were captured using bright field microscopy 7A 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 7B depicts the average pixel number S D in the detection zones for each condition n 8 wells condition A Endpoint Detection of HT 1080 Cell Migration into Detection Zone i 250000 WH N iW i 200000 iy inh i ii I 1 150000 I IN WA ji il HT Hi Wa Wit Wi Hill nil Hi j ST Wii Hi Hi Hit Hil iii TT Hit Ni 1i 100000 Pre Migration Post Migration 50000 t 0 hrs t 24 hrs 0 Pre Migration Post Migration N 8 Condition Figure 7 Cell migration data obtained using Wright Giemsa colorimetric stain T Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0031 04 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 6 Microplate Reader Analysis e Attach the Oris Detection Mask to the bottom of the Oris plate see Step 3 of Protocol e Optimal settings will vary according to the microplate reader mak
9. e 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 Sample data using a fluorescent stain and microplate reader analysis are shown in Figure 8 HT 1080 cells were fluorescently stained with CellTracker Green and wells populated with Oris Cell Seeding Stoppers were seeded with 50 000 cells i e 100 uL of 5x10 cells mL After a 4 hour incubation 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 28 hours and at various time points the fluorescence signals in the detection zones were measured using a microplate reader The images below Figure 8A 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 Figure 8B A Endpoint Detection of HT 108 ar Kinetic Detection of HT 1080 E Cell Migration into Detection Zone if inl Average Pre Migration t 0 hrs Post Migration t 21 hrs 10 15 20 Time hrs n 9 wells time point Figure 8 Cel
10. ell 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 Tool and Oris Cell Seeding Stoppers The Oris Universal Cell Migration Assembly Kit 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 Universal Cell Migration Assembly Kit has been designed for use with adherent cell cultures This assay has been successfully used with 3T3 Swiss albino HT 1080 HCEC HUVEC and MCF10A cell lines Using the Oris Universal Cell Migration Assembly Kit offers the following features amp benefits e Membrane free Cell Migration or Invasion perform e Real time Monitoring monitor changes in cell studies without manipulating transmembrane inserts structure in real time throughout the experiment observe live images of cell movement e Versatile Detection analyze cells treated with multiple e Creative Assay Design coat any ECM or BME on the fluorescent probes labels or stains by using a plate to create a 2 D environment for cell migration or microscope digital imaging system or fluorescence cell invasion assays microplate reader e Preserve Cell Morphology eliminate need for cells to e Reproducible Results obtain greater reproducibility penetrate through a polycarbonate membrane cells can using the Or
11. is Cell Seeding Stoppers compared to move across a treated surface or within a user applied wound healing or scratch assays ECM e Flexible perform kinetic or endpoint assays without the use of special instrumentation Insert Stoppers Allow Cells to Analyze Cells in Seed Cells onto Stoppers to Migrate into Detection Zone Oris Plate amp Create Detection Detection Zone Microplate Reader Allow to Adhere Zone Analysis Detection Mask Attached Image Analysis No Mask Required Figure 1 Schematic of Oris Universal Cell Migration Assembly Kit u Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0031 04 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 2 ll ORIS PLATE DIMENSIONS Diameter of Stopper Space Detection 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 Detection Zone per Well 3 14 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 2 mm Distance between Wells Thickness of Well Bottom Storage Conditions Refrigerate 4 C Important Read Instructions Before Performing any Oris Assay lll MATERIALS PROVIDED Product No CMAU101 Product No CMAU505 Oris compatible 96 well black clear bottom Plate
12. ith 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 Oooo 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 Universal Cell Migration Assembly Protocol and microplate reader settings for analysis of cell migration using a fluorescence microplate reader T Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0031 04 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 9
13. l migration data obtained using CellTracker Green fluorescent stain PLATYPUS Platypus Technologies LLC SP0031 04 5520 Nobel Drive Suite 100 Madison WI 53711 USA www platypustech com Toll Free 866 296 4455 Phone 608 237 1270 Fax 608 237 1271 pg 7 VII ORDERING INFORMATION l l pack PROCMA1 renee Tissue Culture Treated 5 pack PROCMAS vey Biocompatible Gel Cell Migration Assays ESET T 1 pack PROCMACC1 8 5 pack PROCMACCS Tissue Culture Treated 5 pack PRO384CMAS Oris Pro 384 l RIT Biocompatible Gel Cell Migration Assays Collagen I Coated 5 pack PRO384CMACC5 l pack CMA1 101 5 pack CMA5 101 Tissue Culture Treated Collagen I Coated I pack CMACCI 101 Oris Cell Migration 5 pack CMACCS 101 Oris Cell Seeding Stoppers Assays pre populated Fibronectin Coated pack O MARN IU 5 pack CMAFN5 101 l pack CMATR1 101 Universal l pack CMAU101 Oris Cell Migration Tissue Culture Treated 5 pack CMAUS05 Oris Cell Seeding Stoppers Assembly Kits not pre populated FLEX Tissue Culture Treated 4 pack CMAUFL4 Collagen I 1 pack PROIA1 Oris Pro 96 well low overlay conc 3 pack PROIA3 ee as Invasion Assays Collagen I 1 pack PROIAPLUS1 high overlay conc 3 pack PROIAPLUS3 Biocompaunie Oe For a complete list of assays visit Platypus Technologies at www platypustech com order_main html For technical assistance contact Technical Support at 866 2
14. ncentration This procedure is intended to assist in determining the cell seeding density needed to achieve confluency of your cell line when using the Oris Universal Cell Migration Assembly Kit 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 Prepare a log phase culture of the cell line to be tested Collect cells and determine the total number of cells present 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 Dispense 100 uL of cell suspension per well into the 96 well plate to result in the following plate layout Column te 3 Cells well 100 000 50 m 25 m Number of wels 8 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 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
15. of the plate e Lift the Oris Stopper Tool vertically to gently remove the stoppers NOTE DO NOT use the Oris 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 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 Optional If the plate was coated with an ECM in Step 1 an overlay of ECM may be introduced in the wells to facilitate a 3 D Figure 6 Removal of Stoppers Panels A B and C Position Shee ad ae the Tines of the Stopper Tool between the Stopper invasion assay Optimization of experimental conditions will be Tips D Lift Vertically and E Do NOT Pry Stoppers required to establish invasion conditions for a given cell line Add 100 uL of fresh culture media to each well Cells may be examined microscopically throughout the incubation period to monitor progression of migration Migration time will vary depending upon cell type experimental design and ECM If performing an endpoint analysis of cell migration stain cells with a fluorescent stain after sufficient migration has occurred Refer to Section VI and Appendix II for further information on data acquisition and fluorescence staining technique NOTE Oris Cell Seeding Stoppers are for single use only Platypus cannot guarantee
16. perature Optional If desired coat the bottom of the wells with an ECM component collagen fibronectin laminin etc and allow the ECM to dry prior to populating the plate with the Oris Cell Seeding Stoppers 2 Under sterile conditions populate the 96 well plate with Oris Cell Seeding Stoppers 2 AIII Ml Il Il i li INI iu ei ll e 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 e 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 i iii i Il WM Mli Mii Nh wiz O NOTE It is extremely important to ensure that the stoppers g are inserted perpendicular to the well bottom and are fully ea a yourdate cet Mu recurs dats ste with lo eve Floure 2 Stopper serion Process A Placement of Stopper
17. 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 NOTE For best results add or extract media by placing the pipette tip along the wall of the well see Figure 5 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 Figure 5 Media is Added with Pipette 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 Designate several reference wells in which the stoppers will remain in place until results are read t O pre migration controls Using the Oris Stopper Tool remove all other stoppers see Figure 6 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
18. s i i into Wells B Close up of Stoppers Partially Inserted into potential for lt 12 the pre populated Oris Cell Migration Wells C Proper Placement of Stoppers D Pressing of Assay kit CMA1 101 is recommended Stoppers into Wells and E Fully Inserted Stoppers 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 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 Q NOTE the sealing of the stoppers can be most easily observed if the plate is tipped 2 at an angle and viewed under indirect light to reveal the bullseye pattern at the bottom of each well 4 Apply the Oris Detection Mask to the bottom of the 96 well plate if microplate Figure 3 Stoppers that are reader data is being collected The Detection Mask is not necessary if collecting A Partially Sealed imaging data B Unsealed 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 in the wells Aperture Orientation A 1 Corner e Orient the chamfered corners of the mask with those of the
19. s products shall conform substantially to the description of such goods as provided in product catalogues and literature accompanying the goods until their respective expiration dates or if no expiration date is provided for 6 months from the date of receipt of such goods PLATYPUS will replace free of charge any product that does not conform to the specifications This warranty limits PLATYPUS s liability only to the replacement of the nonconforming product THIS WARRANTY IS EXCLUSIVE AND PLATYPUS MAKES NO OTHER WARRANTY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE The stated express warranties and the remedy provided for breach thereof are in lieu of all other liability or obligations of PLATYPUS for any damages whatsoever arising out of or in connection with the delivery use misuse performance or the inability to use any of its products 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 effe
20. the integrity of the stopper material after a second sterilization procedure T Platypus Technologies LLC 5520 Nobel Drive Suite 100 Toll Free 866 296 4455 SP0031 04 Madison WI 53711 USA Phone 608 237 1270 www platypustech com Fax 608 237 1271 pg 5 DATA ACQUISITION The readout of the Oris Universal Cell Migration Assembly Kit can be conducted 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 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 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 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 th
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