Technical Bulletin #436 Set Up Guidelines and Dimensional
Contents
1. Technical Bulletin 436 Set Up Guidelines and Dimensional Templates for Fluorescence Plate Readers used with Corning FluoroBlok Insert Systems and Corning BioCoat Multiwell Insert Cell Based Assays Corning Life Sciences Tewksbury MA Please note unless specified otherwise the data generated in this Technical Bulletin TB used the original purple FluoroBlok membrane We have made improvements to this membrane since it was introduced The new FluoroBlok uses a black membrane with improved spectral characteristics General information in this TB applies to both versions of the product but the specific wavelength ranges mentioned here apply only to the original purple version unless specified otherwise For details see Technical Bulletin CLS DL CC 042 New PET Membrane for Corning FluoroBlok 3 0 um and 8 0 um Pore Size Cell Culture Inserts CORNING Introduction Corning FluoroBlok Inserts and Corning BioCoat Multiwell Insert Cell Based Assays provide platforms for real time analysis of samples using fluorescence based detection These products are used for a variety of applications including analyses of cell motility and compound permeability To monitor the appearance of fluorescence in the chamber located below the insert a bottom reading fluorescence plate reader is required This Technical Bulletin describes set up guidelines for a variety of instruments that are amenable to insert based assa2. Some systems do not support user entered plate maps or plate definitions If you are using such a system the names mentioned for plate choice in this technical bulletin may be subtly different than what is in your software The placement of the insert wells in the 24 Multiwell format is not symmetrical and requires a non standard 24 well plate dimension In some plate readers the individual 24 well or 24 Multiwell Insert plates must be read without the lid e If additional information is needed regarding the reference points and plate reader set up please contact the instrument manufacturer technical support group Corning is not responsible for damaged property associated with defining new plate maps or instrument modification Template Set Up To add a new plate format template to the plate reader template menu enter the plate layout dimensions into the plate reader software formula The required values for some commonly used plate readers are listed in this Technical Bulletin Detailed drawings with exact well locations are available by contacting Technical Support Please consult the instrument User Manual to obtain key reference points and units Corning Life Sciences strongly recommends you familiarize yourself with the plate reader and have the templates loaded in your plate reader prior to starting your experiment Autofluorescence Background If fluorescence is monitored with a top reading instrument the Corning Fluo
3. e Open the Ascent software program Select the Setup menu heading e Open the Filters menu heading drop down list appears e Examine the Excitation and Emission Filter combinations and determine whether the appropriate filters for the fluorophore to be detected have been installed Note If the appropriate filters are not installed refer to the Thermo LabSystems reader s manual for instructions Plate Dimensions DO NOT READ INDIVIDUAL 24 WELL or 24 MULTTIWELL FORMATS WITH THE LID ON THE PLATE To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner e Open the Ascent software program e Select the Setup menu heading e Open the Plate Formats menu heading drop down list appears e Under Setup Plate Templates select the appropriate template for your application e g 24 well Corning FluoroBlok to read Corning FluoroBlok 24 well Individual Insert Systems 24 Multiwell Corning FluoroBlok to read 24 Multiwell System etc Note The standard pre set parameters for 24 wells Corning 3047 is similar to that for Corning FluoroBlok Individual Inserts and will work acceptably with the individual insert plates If the correct plate map is on the list select it and continue To verify the installed settings are correct do the following e Select the Modify box to view parameters e Check the parameters against the data in Table 7 e If any parameters are incorrec
4. from the drop down menu e Reads per well 24 well plates 4 e Reads per well 96 well format 1 e Center Only Gain 55 e Probe Position The probe must be in the bottom read position The probe position is not software switchable it must be manually repositioned Repositioning the probe is a brief operation Please refer to the Applied Biosystems CytoFluor manual for specific instructions Table 1 Corning FluoroBlok Corning FluoroBlok Corning FluoroBlok Cell Culture Insert 24 Multiwell Insert 96 Multiwell Insert Rows Columns X1 X2 Y1 Y2 Bio Tek Synergy Set Up Prior to reading the insert plate determine that the reader has the appropriate Excitation and Emission Filter set installed and the proper insert plate type is specified in the Plate Format list Check Installed Filter Set e Open the KC4 software program e Select Wizard e Select Filter Set e Select the appropriate Excitation and Emission settings for your fluorophore e Select Sensitivity Note Sensitivity will have to be optimized for your specific application A setting of 50 is a good starting point Auto gain is not recommended Plate Dimensions To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner e Select System e Select Plate Formats Select appropriate plate type e g Corning FluoroBlok Individual Corning FluoroBlok 24 Multiwell or Corning FluoroBlok
5. tab and examine the list for the available Corning FluoroBlok Insert Plate formats If the correct plate type is on the list select it and continue If it is not found create a new template as follows e From the Plate Types Screen select the Add button e Enter plate type name e g Corning FluoroBlok 24 Multiwell or Corning FluoroBlok 96 Multiwell Click OK e On the Plate Properties screen enter the appropriate plate parameters for the insert plate as shown in Table 4 Click OK Table 4 Corning FluoroBlok Corning FluoroBlok Corning FluoroBlok Cell Culture Insert 24 Multiwell Insert 96 Multiwell Insert Number of Wells in Row 4 4 8 Number of Wells in Column 6 6 12 Unlidded Plate Height 21 225 19 05 Plate Height with Cover 23 4 24 4 19 3 Strip Orientation Horizontal Horizontal Horizontal Edge of Plate to Well Center Horizontal 13 75 12 7 15 38 Edge of Plate to Well Center Vertical 13 75 13 75 11 01 Well to Well Horizontal 19 3 19 3 8 996 Well to Well Vertical 19 3 19 3 8 997 continued PerkinElmer Victor continued Entering a Protocol Open the Victor software program Select the Protocol Explorer menu bar icon Right click the Users Folder Select New Protocol Group and enter a name for the new folder Click OK Right click the new folder and select New Protocol Enter the protocol name Click OK Double click the new protocol Select the Plate tab and enter the following settings Measurem
6. 96 Multiwell from the pull down menu If the list does not contain a plate with the correct dimensions a new plate can be defined as follows e Select New e Enter the template information in the Plate Description dialog box as shown in Table 2 e Select wells to be read e Click Next until the end e Click OK e Save protocol e Select New for each new plate Select read for each new read on the same plate Table 2 Length Width Top Left X Top Left Y Bottom Right X Bottom Right Y Columns Rows Well Diameter Height Corning FluoroBlok Cell Culture Insert 127640 85470 14020 13780 110540 71690 6 4 Corning FluoroBlok 24 Multiwell Insert 127640 85470 12970 13780 109490 71690 6 4 Corning FluoroBlok 96 Multiwell Insert 127760 85470 14100 11520 113080 74510 12 8 3180 19300 BMG FLUOstar Galaxy and OPTIMA Set Up Prior to reading the insert plate determine that the reader has the appropriate Excitation and Emission Filter set installed and the proper insert plate type is specified in the Plate Type list To accommodate the height of the insert plate spacers are needed to raise the optics above the insert plate surface To obtain spaces please call 877 BMG LABS and request BMG Cat No 11 701 Note The Galaxy and OPTIMA use different units for entering template data Reader Configuration e Open the FLUOstar software program e Select the Setup icon on the menu ba
7. Multiwell formats as they are too tall to fit in the reader Set Up Prior to reading the insert plate determine that the reader has the appropriate Excitation and Emission Filter set installed and the proper insert plate type is specified in the Plate Type list Check Installed Filter Set e Open the CytoFluor software program e Open the Excitation and Emission Filter drop down menus and choose the appropriate filter set Note If the appropriate filters are not installed refer to the CytoFluor manual for instructions Plate Dimensions DO NOT READ INDIVIDUAL 24 WELL or 24 MULTIWELL FORMATS WITH THE LID ON THE PLATE To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner e Open the CytoFluor software program e Open the Plate Type drop down menu and examine the list for the available Corning FluoroBlok Insert Plate format If the correct plate type is listed select it and continue If it is not found create a new template as follows e Select Plate on the menu bar e Select Define Plate Enter the plate type name in the box e g Corning FluoroBlok 24 Multiwell or Corning FluoroBlok 96 Multiwell e Enter the appropriate plate parameters for the insert plate format as shown in Table 1 Select Add and OK Complete Set Up e Use default settings except as noted below Select the Plate Type from the drop down menu Select the Excitation and Emission Filter
8. ces e Under the General tab in the drop down menu select Fluorescence detection mode e From the drop down menu select the appropriate plate definition from the Plate tab if desired check the Multiple reads per well box then select a pattern e g square and number of replicates e g 2 x 2 from the available options Select the appropriate Excitation and Emission filters from the Meas Params tab drop down menu also select Bottom as Read mode choose a Gain setting method manual optimal or from a specific well and use default integration parameters zero time lag 40 psec integration time e Close the Edit Measurement Parameter menu item then select the Start Measurement menu item Table 6 Corning FluoroBlok Corning FluoroBlok Corning FluoroBlok Cell Culture Insert 24 Multiwell Insert 96 Multiwell Insert Columns 6 6 12 Rows 4 4 8 Well Form Round Round Round Well Diameter 6 4 6 5 3 18 Upper Left Well X 265 X 1560 X 450 Start Position Y 3000 Y 2812 Y 375 Lower Right Well X 95935 X 94825 X 98895 End Position Y 60750 Y 60937 Y 63000 Unlidded Plate Height 21000 um 22500 um 17020 um Plate Height with Cover 23400 um 24500 um 19300 um Thermo LabSystems Fluoroskan Ascent Set Up Prior to reading the insert plate determine that the reader has the appropriate Excitation and Emission Filter set installed and the proper insert plate type is specified in the Plate Format list Check Installed Filter Set
9. dth 854 854 855 X 1 140 130 141 Y 1 138 138 115 X N 1105 1095 1131 Y N 717 717 745 Format 24 24 96 Table 3B OPTIMA Corning FluoroBlok Cell Culture Insert Length 127 50 Width 85 40 X 1 14 00 Y 1 13 80 X N 110 50 Y N 71 70 Format 24 Corning FluoroBlok 24 Multiwell Insert 127 50 85 40 13 00 13 80 109 50 71 70 24 Corning FluoroBlok 96 Multiwell Insert 127 80 85 50 14 10 11 50 113 10 74 50 96 PerkinElmer Victor Set Up Prior to reading the insert plate determine that the reader has the appropriate Excitation and Emission Filter set installed and the proper insert plate type is specified in the Plate Type list Check Installed Filter Set e Open the Victor software program e Select Tools on the menu bar Select Filters from the drop down menu Note If Filters is grayed out then select User Level then click Advanced Select the Emissions Filters tab and examine the list for the appropriate filter Select the CW Lamp Excitation Filters tab and examine for the appropriate filter Note If the appropriate filter set is not installed please refer to the Victor manual for instructions Plate Dimensions To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner e Open the Victor software program e Select Tools on the menu bar e Select Miscellaneous Settings on the drop down menu e Open the Plate Types
10. ent height standard Plate Type Select appropriate plate Individual 24 Multiwell 96 Multiwell Select the Measurement tab and enter the following settings Measurement Mode By plate Click the Labels icon button If the correct label parameters are already entered select it and click OK If not enter the parameters as follows Select the Fluorometry tab Select the Add button Enter a new label name e g Corning FluoroBlok 24 Multiwell Click OK On the Fluorometry Label Properties screen enter the following parameters e CW Lamp Energy Scale slider at mid point e CW Lamp Filter select from drop down menu e Emission Filter select from drop down menu e Emission Aperture Normal e Counter Position Bottom e Counter Height 0 1 Click OK Select File on the menu bar and Save the protocol file e To start a protocol select Start Note If a window with the following error appears Wallac 1420 Exception PLATE ERROR AT MEASUREMENT UNIT The plate is lower than described in the protocol click Ignore PerkinElmer HTS 7000 Set Up Prior to reading the insert plate determine that the reader has the appropriate Excitation and Emission Filter set installed and the proper insert plate type is specified in the Plate Format list Check Installed Filter Set e Open the HTSoft software program e Select Cancel if the HTSoft Wizard window appears e Open the Instrument menu tab and select Parameter Setup Se
11. er to the SpectraFluor Plus manual for instructions Plate Dimensions DO NOT READ INDIVIDUAL 24 WELL or 24 MULTIWELL FORMATS WITH THE LID ON THE PLATE To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner e Open the Xfluor4 xls software program enable macros when requested e Open the Xfluor4 menu tab Set all operational parameters Table 6 e Open the Plate tab drop down menu and select Browse to examine the list of available Plate Definition Files pdf Select the appropriate Corning Insert System plate type If the correct plate type is on the list select it and continue If it is not found create a new template as follows e Exit Plate Definition File list and return to main Excel screen e Open the Xfluor4 menu tab again then select Edit PlateDefinition tab e Enter the appropriate plate parameters for the insert plate format as shown in Table 6 e Click Update then click Close this does not save the file Under File menu select Save PlateDef as and save the Plate Definition File e g Corning FluoroBlok 24 Multiwell or Corning FluoroBlok Individual Reading Samples Using Fluorescence Based Detection e Open the Xfluor4 xls software Enable macros when requested Connect to reader e In the Xfluor4 menu list open the Edit Measurement Parameter menu item this opens a tabular listing of available choi
12. lect the General tab and under Detection Method select Fluorescence mode Select the Measurement tab and check and or set Excitation and Emission Filters inspect the drop down menus under Excitation and Emission to examine each list for the appropriate filter set Note If the appropriate filters are not installed refer to the HTS 7000 manual for instructions Plate Dimensions DO NOT READ INDIVIDUAL 24 WELL or 24 MULTIWELL FORMATS WITH THE LID ON THE PLATE To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner To see if the plate is already defined e Select the Instrument tab e Select Parameter Setup e Select the General tab e Browse the Plate Definition file If the plate definition file is not found create a new definition as follows e Close the Instrument Parameter window e Under File menu select HTSoft Wizard e Select New Plate Definition e Open the File menu tab and select Open PlateDef and select a plate with similar number of wells Close the window Under the File menu select Edit PlateDef e Enter the parameters as shown in Table 5 e Select the File menu tab select Save PlateDef as and enter the new name e Select Exit Reading Samples Using Fluorescence Based Detection e Open the HTSoft software program e Under File select HTSoft Wizard in the drop down menu e Select Create and Save a New Method e Select Next e Select the Measuremen
13. orner Well Distance Y 630 Well Type Circle Can be read with Lid No Note Gain settings are automatically set by the plate reader Corning acquired the BioCoat and FluoroBlok brands For information visit www corning com discoverylabware For Research Use Only Not intended for use in diagnostic or therapeutic procedures For a listing of trademarks visit us at www corning com lifesciences trademarks All other trademarks are property of their respective owners Corning Incorporated One Riverfront Plaza Corning NY 14831 0001 CORNING Corning Incorporated Life Sciences 836 North St Building 300 Suite 3401 Tewksbury MA 01876 t 800 492 1110 t 978 442 2200 f 978 442 2476 www corning com lifesciences 2012 2013 Corning Incorporated Printed in USA 3 13 CLS DL CC 074
14. r to open a drop down menu e Select Reader Configuration Select Fluorescence Intensity and Time Resolved Fluorescence e Click OK Note You will be prompted to check that the right measurement head is installed The correct head can be identified by the presence of two yellow dots on this surface Check Installed Filter Set e Select the Setup icon on the menu bar to open a drop down menu Select Filters e Examine the list for the appropriate filters If the appropriate filters are not listed refer to the FLUOstar manual for instructions e Click OK Plate Dimensions To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner Select the Setup icon on the menu bar to open a drop down menu e Select Microplates e Examine the list for a defined plate with the correct dimensions as shown in Table 3A Galaxy or 3B OPTIMA If the list does not contain a plate with the correct dimensions a new plate can be defined as follows Select New e A new window will open Enter the dimensions for the insert plate format as shown in Table 3A Galaxy or 3B OPTIMA e Click OK Bottom Read Optics Turn both optics positioning wheels so that the first position of each is located at 12 o clock Table 3A Galaxy Corning FluoroBlok Corning FluoroBlok Cell Culture Insert Corning FluoroBlok 24 Multiwell Insert 96 Multiwell Insert Length 1275 1275 1278 Wi
15. roBlok PET membrane exhibits negligible autofluorescence across the useful range spectrum 490 700 nm However a low level of background may be detected with a bottom reading instrument due to autofluorescence and or a reflection from the polystyrene base plate The use of high gain settings lamp energy or other terms may be used or the lack of appropriate assay controls may promote an auto fluorescence effect that is independent of insert mediated autofluorescence A gain setting that is too high may also lead to saturation of the detector with samples that exhibit very high fluorescence The optimal gain or lamp intensity settings must be determined empirically As a starting point initiate the experiment with a gain setting or lamp intensity setting at the midpoint Fluorescence Detection Issues Note Prior to reading Corning FluoroBlok Inserts or Corning BioCoat Multiwell Insert Cell Based Assays ensure that the reader has the appropriate Excitation and Emission Filter set installed Appropriate Excitation and Emission Filters for detection of fluorophore s used in cell labeling must be employed unless a monochromator based plate reader e g Tecan Safire is available To ensure that all samples are measured as accurately as possible an appropriate gain or lamp intensity setting must be used Applied Biosystems CytoFluor 4000 Note The CytoFluor 4000 TC Temperature Control model cannot read the 24 well or 24
16. t edit them and save the edited Plate Format definition Template parameter dimensions are in units of 1 10 mm 100 microns e Click OK several times to save the new Plate Format and return to the main menu e Verify the correct parameters were entered and saved continued Thermo La bSystems If the correct plate map is not listed you can create a new template as follows e Select a similar but unused plate map You should select a 24 well template for Fluoroskan Ascent the 24 Multiwell Insert System and a 96 well template for the 96 Multiwell Insert continued System e Select the Duplicate box e Rename the template e g Corning FluoroBlok 24 wells to read Corning FluoroBlok 24 well Individual Inserts or Corning FluoroBlok 24 Multiwell to read 24 Multiwell System etc Select the Modify box e Enter the appropriate plate parameters as shown in Table 7 Template parameter dimensions are in units of 1 10 mm 100 microns e Click OK several times to save the new Plate Format and return to the main menu e Verify the correct parameters were entered and saved Table 7 Corning FluoroBlok Corning FluoroBlok Corning FluoroBlok Cell Culture Insert 24 Multiwell Insert 96 Multiwell Insert Plate Size X 1275 1275 1278 Plate Size Y 854 854 855 Plate Height 210 without lid 225 without lid 193 Count X 6 6 12 Count Y 4 4 8 Diameter X 64 64 31 Diameter Y 64 64 31 Start X 141 Start Y TS Corner Well Distance X 990 C
17. t Parameters tab e Under the General tab select the Fluorescence Detection Mode and the Plate Definition created above e Under the Measurement tab select the appropriate Filter settings and bottom reading e Complete the Method and save with a new method name e Within HTSoft Wizard select Run an existing method Table 5 Corning FluoroBlok Corning FluoroBlok Corning FluoroBlok Cell Culture Insert 24 Multiwell Insert 96 Multiwell Insert Columns 6 6 12 Rows 4 4 8 Well Form Round Round Round Well Diameter 6 4 6 5 3 18 Upper Left Well X 265 X 1560 X 450 Start Position Y 3000 Y 2812 Y 375 Lower Right Well X 95935 X 94825 X 98895 End Position Y 60750 Y 60937 Y 63000 Unlidded Plate Height 21000 um 22500 um 17020 um Plate Height with Cover 23400 um 24500 um 19300 um Tecan SpectraFluor Plus Set Up Prior to reading the insert plate determine that the reader has the appropriate Excitation and Emission Filter set installed and the proper insert plate type is specified in the Plate Type list Check Installed Filter Set e Open the Xfluor4 xls software program enable macros when requested e Open the Xfluor4 menu tab to set all operational parameters Table 6 e Open the Edit Measurement Parameter tab to check and or set Excitation and Emission Filters inspect the drop down menus under Excitation and Emission and examine each list for the appropriate filter set Note If the appropriate filters are not installed ref
18. ys To determine the optimal set up parameters we performed fluorescence based assays using Corning FluoroBlok inserts in conjunction with a number of fluorescence plate readers including the Applied Biosystems CytoFluor 4000 Bio Tek Synergy BMG FLUOstar Galaxy and OPTIMA PerkinElmer Victor PerkinElmer HTS 7000 Tecan SpectraFluor Plus and Thermo LabSystems Fluoroskan Ascent Important The information contained within this Technical Bulletin applies only to Corning FluoroBlok Insert Systems and Corning BioCoat Multiwell Insert Cell Based Assays i e Tumor Invasion Systems Angiogenesis Systems Insert System Assembly and Orientation To properly orient the insert plate in the instrument the well labeled A1 should be located at the top left corner With the plate in this position the Corning logo will be located on the right side Plate Reader Set Up For use with Corning FluoroBlok Inserts the fluorescence plate reader must support bottom reading fluorescence detection i e excitation light is presented to the sample through the bottom of the base plate and emitted light is collected from the bottom If top and bottom reading are supported by the instrument in use one typically may switch between reading modes by software control or manual reconfiguration of the hardware Before proceeding ensure that the bottom reading mode is operative and or specified by the stored plate reading method if applicable Notes e
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