Radius™ 96-Well Cell Migration Assay
Contents
1. Product Manual Radius 96 Well Cell Migration Assay Catalog Number CBA 126 96 assays CBA 126 5 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Cell migration is a highly integrated multistep process that orchestrates embryonic morphogenesis tissue repair and regeneration It plays a pivotal role in the disease progression of cancer mental retardation atherosclerosis and arthritis The initial response of a cell to a migration promoting agent is to polarize and extend protrusions in the direction of the attractant these protrusions can consist of large broad lamellipodia or spike like filopodia In either case these protrusions are driven by actin polymerization and can be stabilized by extracellular matrix ECM adhesion or cell cell interactions via transmembrane receptors The Radius Cell Migration Assay Kit utilizes a proprietary 96 well plate to monitor the migratory properties of cells Each plate well contains a 0 68 mm non toxic biocompatible hydrogel spot Radius Gel where cells cannot attach When adherent cells are seeded in the Radius Cell Migration well they attach outside of the Radius Gel coated area Once firm cell attachment is achieved the hydrogel is quickly removed to expose a cell free region to study cell migration closure This format provides a robust in vitro system to measure 2 D c2. add 100 uL of Radius Wash Solution to each well Proceed to the Cell Seeding Section below Note Wells can remain in the Radius Wash Solution for up to 1 hour II Cell Seeding l Harvest and resuspend cells in culture medium at 0 1 0 3 x 10 cells ml Note Cell seeding density is highly cell line dependant factoring in cell size spreading and division Ideally the desired monolayer confluency at the start of migration after Radius Gel Removal step should be 80 90 Optimization is recommended and can be done in a standard 96 well cell culture plate prior to migration assays Carefully aspirate the Radius Wash Solution from the wells step 5 above Do not allow wells to dry Note Avoid potential damage to the Radius Gel Spot located in the center by aspirating from the edge of the well Slowly add 100 uL of the cell suspension to each well by carefully pipetting down the wall of the well Transfer the plate to a cell culture incubator for 4 24 hours to allow firm attachment spreading Take care to avoid shaking or bumping the plate HI Radius Gel Removal l 2 Carefully remove the Radius Migration Plate from cell culture incubator Aspirate the media from each well and wash 3 times with 100 uL of fresh media Do not allow wells to dry Note These washes are intended to remove debris or any dead unattached cells Prepare sufficient 1X Radius Gel Removal Solution for all wells by dilutin
3. cells were seeded at 20 000 cells well overnight After Radius Gel removal the cells were allowed to migrate for 24 hrs in the presence of various Cytochalasin D concentrations 10 J CELL BIOLABS INC A a References 1 Ridley AJ Schwartz MA Burridge K Firtel RA Ginsberg MH Borisy G Parsons JT Horwitz AR 2003 Science 302 1704 9 Horwitz R Webb D 2003 Curr Biol 13 R756 9 3 Lauffenburger DA Horwitz AF 1996 Cell 84 359 369 Recent Citations 1 2 Camacho M et al 2015 Prostacyclin synthase expression in head and neck carcinoma patients and its prognostic value in the response to radiotherapy J Pathol 235 125 135 Woodard G E et al 2014 Characterization of discrete subpopulations of progenitor cells in traumatic human extremity wounds PLoS One 9 e114318 Felthaus O et al 2014 Migration of human dental follicle cells in vitro J Periodontal Res 49 205 212 Wong B et al 2013 Adrenomedullin enhances invasion of human extravillous cytotrophoblast derived cell lines by regulation of urokinase plasminogen activator expression and S nitrosylation Biol Reprod 88 34 Ichikawa A et al 2013 CXCL10 CXCR3 enhances the development of neutrophil mediated fulminant lung injury of viral and nonviral origin Am J Respir Crit Care Med 187 65 77 Coulouarn C et al 2012 Hepatocyte stellate cell cross talk in the liver engenders a permissive inflammatory microenviro
4. Reagents 1X Radius Gel Removal Solution Just prior to use prepare a 1X Radius Gel Removal Solution by diluting the provided 100X stock 1 100 in complete culture medium 1X DAPI Fluorescence Stain Just prior to use prepare a 1X DAPI Fluorescence Stain by diluting the provided 1000X stock 1 1000 in PBS Assay Protocol Must be under sterile conditions I Pretreatment of Radius Migration Plate 1 Under sterile conditions remove the Radius 96 well Cell Migration Plate from its packaging Note If wells of the plate have already been used for a previous experiment the unused wells must be completely moisture free before proceeding Please allow the covered plate to dry at room temperature for 1 hour Each plate can handle up to 3 migration experiment cycles 2 Determine which wells will be assayed it is recommended that all samples be tested in triplicate Slowly add 100 uL of Radius Gel Pretreatment Solution to each well by carefully pipetting down the wall of the well Note The Radius Gel Pretreatment Solution should not be added to any wells that will not be used immediately 3 Cover the plate and incubate at room temperature for 20 minutes 4 Carefully aspirate the Radius Gel Pretreatment Solution from the wells Do not allow wells to dry Note Avoid potential damage to the Radius Gel Spot located in the center by aspirating from the edge of the well 5 JaN CELL BIOLABS INC A a 5 Slowly
5. ell migration screen potential inhibitors and study cytoskeleton reorganization events Additional features of the Radius Cell Migration Assay e Exclusive coating method which produces consistent gel spot size 0 68 mm diameter 0 014 mm 2 e Qualitative quantitative real time or endpoint analysis Radius Cell Migration Plate does not need to be used all at once unused wells can be used in future experiments up to a total of 3 migration experiment cycles Compatible with all cell stains dyes and labels Complete migration zone closure achievable in 15 30 hours actual time is cell line dependent Analyze by phase contrast or fluorescence microscopy Compatible with HCS HCI instrumentation Adaptable to liquid handling equipment Optimized for 10X magnification entire Radius Gel migration area is viewed in a 10X magnification field e Radius Gel removal is controlled and extremely fast migration starts almost simultaneously between wells The Radius Cell Migration Assay Kit is designed for High Content Analysis applications or imaging software and is adaptable to liquid handling equipment Each kit provides sufficient quantities to perform 96 migration tests 2 AN CELL BIOLABS INC S a Assay Principle Pretreat desired wells with Radius Gel Pretreatment Solution i Each well contains one 0 68 mm Radius Gel spot above image artificially colored blue Add cell suspension to the w
6. ells and allow 4 24 hours for firm attachment spreading Cells don t attach in the Radius Biocompatible Gel Layer area Remove the Radius Biocompatible Gel Layer exposing the cell free area for cell migration Brightfield Cell Stain Solution Calcein AM y HCS Analysis rt Migration Migration Inhibitor Inhibitor CELL BIOLABS INC Creating Solutions for Life Science Research Related Products ON ee ee E CBA 100 CytoSelect 24 Well Cell Migration Assay Boyden Chamber Assay Colorimetric CBA 101 CytoSelect 24 Well Cell Migration Assay Boyden Chamber Assay Fluorometric CBA 106 CytoSelect 96 Well Cell Migration Assay Boyden Chamber Assay Fluorometric CBA 110 CytoSelect 24 Well Cell Invasion Assay Boyden Chamber Assay Colorimetric CBA 120 CytoSelect 24 Well Wound Healing Assay CBA 130 CytoSelect 96 Well Cell Transformation Assay Soft Agar Colony Formation Radius 96 well Cell Migration Plate Dimensions Well Volume 360 uL Well Depth 10 67 mm Well Diameter Top 6 86 mm Well Diameter Bottom 6 35 mm Plate Length 127 8 mm Plate Width 85 5 mm Plate Height 14 2 mm Al Row Offset 11 2 mm A1 Column Offset 14 3 mm Well Center to Well Center Spacing 9mm Flange or Skirt Height 6 096 mm Well Bottom Elevation 3 57 mm Well Bottom Thickness 0 5 mm Well Bottom Area 0 3165 cm Kit Components l AOD O
7. g the stock 1 100 in culture medium See Preparation of Reagents Section Aspirate the media from the wells and add 100 uL of 1X Radius Gel Removal Solution 5 Transfer the plate to a cell culture incubator for 30 minutes to allow complete gel removal 10 Aspirate the 1X Radius Gel Removal Solution from each well and wash 3 times with 100 uL mL of fresh media Do not allow wells to dry After the final washing is complete add 200 uL of complete medium to each well Agents that inhibit or stimulate cell migration may also be added directly to the wells At this point pre migration images may be captured with an inverted microscope imaging software or HCI HCS instrument Transfer the plate back to the cell culture incubator microscope stage incubator during the migration process Monitor the migration closure by endpoint or real time analysis For time course experiments live cell compatible dyes or labels are required e g Calcein AM GFP RFP For endpoint experiments fixed cell detection should be used Cell Stain DAPI TRITC phalloidin 6 4 CELL BIOLABS INC N ZA IV Optional DAPI Fluorescence Labeling 1 Aspirate the media from the wells and add 100 uL mL of Fixation Solution to each Allow the cells to fix for 10 minutes at room temperature Aspirate and discard the solution Carefully wash each well 3 times with 200 uL of PBS Prepare sufficient 1X DAPI Fluorescence Stain for all wells by dilut
8. ing the stock 1 1000 in PBS See Preparation of Reagents Section Add 100 uL of 1X DAPI Stain to each well to be stained Incubate 15 minutes at room temperature Carefully wash each well 3 times with 200 uL of PBS Add 200 uL PBS to each well to keep cells hydrated Examine wells under an inverted fluorescence microscope with DAPI filter 350nm 470nm PY Ss Oe Soe pn V Optional Cell Staining 1 Aspirate the media from the wells and add 100 uL of Cell Stain Solution to each 2 Allow the cells to stain for 15 minutes at room temperature Aspirate and discard the solution 3 Carefully was each well 3 times with 200 uL of deionized water 4 Discard all washes and allow wells to dry at room temperature 5 Examine wells under an inverted light microscope Analysis of Results There are a number of software programs available for the analysis of cell migration images One of these is CellProfiler Cell Image Analysis Software offered free of charge by the Broad Institute You may find more information on this program online at www cellprofiler org In order to analyze data from our Radius Cell Migration Assays the CellProfiler software must be customized For your convenience we have developed add ons that will customize the program for you Please visit our website at www cellbiolabs com type Cellprofiler in the Search box and follow the instructions to download the appropriate add on CellProfiler i
9. nment that drives progression in hepatocellular carcinoma Cancer Res 72 2533 2542 Alcolea S et al 2012 Interaction between head and neck squamous cell carcinoma cells and fibroblasts in the biosynthesis of PGE2 J Lipid Res 53 630 642 Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2013 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 11 Ja _ CELL BIOLABS INC AEN
10. o po Radius 96 well Cell Migration Plate Part No 112601 One 96 well black walled clear bottom tissue culture treated plate with each well containing one Radius non toxic biocompatible hydrogel spot 96 gel spots total per plate Radius Gel Pretreatment Solution Part No 112502 One Sterile Bottle 13 0 mL Radius Wash Solution Part No 112503 One Sterile Bottle 13 0 mL Radius Gel Removal Solution 100X Part No 112504 One Sterile Tube 150 uL DAPI Fluorescence Stain 1000X Part No 112002 One Amber Tube 30 uL Fixation Solution Part No 122402 One Bottle 20 0 mL Cell Stain Solution Part No 112505 One Bottle 12 0 mL AN 5 CELL BIOLABS INC JE a Materials Not Supplied Oe OA A ee oS Adherent migratory cell lines and culture medium Multichannel pipette Cell culture incubator 37 C 5 CO atmosphere Inverted light microscope with a digital camera Optional Microscope stage or cage incubator Optional Inverted fluorescence microscope with DAPI filter 350nm 470nm Optional Imaging Software for measuring cell migration Optional HCS HCI Instrument Storage Upon receipt aliquot and store the Radius Gel Removal Solution and DAPI Fluorescence Stain at 20 C avoid multiple freeze thaw cycles and transfer the Fixation Solution to 4 C All other kit components should be stored at room temperature until the kit s expiration date Preparation of
11. s a trademark of the Broad Institute There is no relationship between Cell Biolabs Inc and the Broad Institute Cell Biolabs offers these add ons as a courtesy to our customers who wish to analyze data obtained using our Radius Cell Migration Assays CELL BIOLABS INC Example of Results The following figures demonstrate typical results with the Radius 96 well Cell Migration Assay Kit One should use the data below for reference only This data should not be used to interpret actual results aal We ote i Yyy ee ys 4 K gt Py ar KIN r La Figure 1 Various Detection Methods with Radius Cell Migration Assay HeLa cells were seeded overnight After Radius Gel removal cells were stained with DAPI Cell Stain Solution and Calcein AM not included in kit according to the assay protocol HT1080 NIH3T3 4hr 21 hr Figure 2 Cell Migration Time course HT1080 and NIH3T3 cells were seeded at 20 000 cells well overnight After Radius Gel removal the cells were allowed to migrate for various times CELL BIOLABS INC Creating Solutions for Life Science Research Pre Migration 0 031 uM 100 100 80 p 75 s 3 60 S 50 2 9 40 o o 25 a 0 T T 0 T T 0 00 0 01 0 10 1 00 0 00 0 01 0 10 1 00 Cytochalasin D uM Cytochalasin D uM Figure 3 Inhibition of HT1080 Cell Migration by Cytochalasin D HT1080
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