Molecular Devices FlexStation II Operator'S Manual Download

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3 Operating

Procedures

Part 3 explains how to start up the system and how to use the control panel and the
SOFTmax PRO application software to configure instrument settings, define experiment
protocols, and run the analysis, as described in the following sections.

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3.1: Overview

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3.2: Starting Up the System

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3.3: Setting the Temperature

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3.4: Setting Up the Software

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3.5: Configuring Instrument Settings in Flex Mode

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3.6: Other Software Settings

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3.7: Reading the Microplate

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3.8: Shutdown

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3.9: Other Read Modes

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3.10: Optimizing Fluorescence Assays in Spectrum Mode

Note:

Information in part 3 assumes the instrument and computer are installed and

connected properly. For further information, see part 2, Installation Procedures.

FlexStation II Operator’s Manual – Rev. D

Summary of Contents for FlexStation II

Page 1: ...FlexStation II and FlexStation II384 Benchtop Scanning Fluorometer and Integrated Fluid Transfer Workstation Operator s Manual PN 0112 0077 Rev D ...

Page 2: ...le California 94089 United States of America Patents The FlexStation instrument and methods have US and International patents pending Trademarks SPECTRAplate and AUTOMIX are trademarks and FlexStation and SOFTmax are registered trademarks of Molecular Devices Corporation Cliniplate is a registered trademark of Labsystems DELFIA is a registered trademark of Wallac Oy Emerald II is a trademark of Tr...

Page 3: ...to Fluorometry 19 1 6 2 Instrument Design 21 1 6 3 Modes of Operation 24 1 7 Introduction to SOFTmax PRO Application Software 27 2 Installation Procedures 31 2 1 General Precautionary Information 31 2 2 Unpacking the System 32 2 3 Installing the Fluidics Module 35 2 4 Installing the Pipettor Head 37 2 5 Setting Up the Computer 40 2 6 Connecting the Cables 41 2 7 Installing the Drawer Adapters 42 2...

Page 4: ...7 3 5 11 Pipette Tips Layout 68 3 5 12 Compound and Tip Columns 69 3 5 13 AutoRead 72 3 5 14 Pipette Tip Air Gap 72 3 5 15 Settings Displayed in Plate Sections 73 3 6 Other Software Settings 74 3 7 Reading the Microplate 75 3 8 Shutdown 78 3 9 Other Read Modes 78 3 10 Optimizing Fluorescence Assays in Spectrum Mode 79 4 Applications 85 5 Service and Maintenance Procedures 87 5 1 Technical Support ...

Page 5: ... a Mechanical Problem 103 6 4 2 Opening the Instrument 104 6 4 3 Evaluating the Tip Rack 105 6 4 4 Inspecting Inside the Fluidics Module 106 6 4 5 Removing the Pipettor Head 109 6 4 6 Expelling Undispensed Fluid from Tips 111 6 4 7 Recovery Procedure 112 6 5 General Error Messages 113 6 6 Tilting or Removing the Fluidics Module 115 6 6 1 Tilting the Fluidics Module 116 6 6 2 Removing the Fluidics ...

Page 6: ...FlexStation II Operator s Manual Rev D vi ...

Page 7: ...tes The instruments offer 96 and 384 well microplate to microplate fluid transfer 8 or 16 wells at a time and can read microplates having 6 12 24 48 96 and 384 well formats Top or bottom detection is available with a simple click of a button A microplate adapter is required in the reading chamber drawer for 96 and 384 well assay plates for both bottom and top reading The black adapter allows for o...

Page 8: ...s the incubator temperature from 1 C above ambient to 45 C The system operates in one integrated fluidics read mode and four read only modes Using the Automix feature the contents of the wells in a microplate can be mixed automatically by shaking which makes it possible to perform kinetic analysis of solid phase enzyme mediated reactions such as a kinetic ELISA In Flex mode the pipettor can also b...

Page 9: ...ex fluidics read mode Part 4 Applications provides a space for detailed protocols for several FlexStation II mode experiments Part 5 Service and Maintenance Procedures provide instructions for routine mainte nance including replacing the lamp changing the pipettor head moving the system to a new location and minor cleaning Part 6 Troubleshooting Procedures provide instructions for diagnosing and s...

Page 10: ...l and on system labels If you use the system in a manner not specified by Molecular Devices any protections provided by the system may be impaired Service Trained Users There are two types of users described in this manual Most procedures required for operating and troubleshooting can be performed by any user who has read the instructions in this manual and is familiar with the system However all ...

Page 11: ...l Support Replacement fuses Use replacement fuses with the required current rating and specification Improper fuses or short circuiting the fuse holders can cause fire or damage the instrument Power rating Ensure the system is connected to a power receptacle that provides voltage and current within the specified rating for the system Use of an incompatible power receptacle can create electrical sh...

Page 12: ...s 1 4 3 Fluidics Module 1 4 4 Detection Module 1 4 5 Computer 1 4 6 Accessories 1 4 7 Consumables actual C set pt C Set Temp Temp on off Drawers reading chamber compounds tip rack Instrument Front View FLEXstation 100 240V 5A 50 60 Hz T6 3 AH s n FS 01001 C NRT L C LR88 026 PATENTS PENDING US Instrument Rear View 6 FlexStation II Operator s Manual Rev D ...

Page 13: ...l C set pt C Set Temp Temp on off Drawers reading chamber compounds tip rack Lifting Off the Top Cover Control Panel The control panel consists of an LCD and six pressure sensitive membrane keys which can be used to initiate and regulate the temperature and to open close the drawers A 2 3 character liquid crystal display LCD shows the current instrument temperature at all times and the set point t...

Page 14: ...er cable and power cords to the instrument FLEXstation 100 240V 5A 50 60 Hz T6 3 AH s n FS 01001 C NRTL C LR88026 PATENTS PENDING US Parallel Port not currently in use RS 232 Serial Port Power Cord Receptacle RS 232 Serial Cable Power Cord Fuse Box Cover Power Switch Input Output Panels Lamp Cover The plastic lamp cover provides access to the flash lamp on the right side of the instrument as viewe...

Page 15: ...chamber compounds tip rack Tip Rack Drawer Compound Plate Drawer Reading Chamber Drawer Instrument with Drawers Open and Carriages Accessible Small plastic pushers located in the front left corner of each drawer hold the plates racks or reservoirs securely in place when the drawers are closed Carrier Pusher Drawer Detail Caution Do not obstruct the movement of any of the drawers If you must retrie...

Page 16: ...und plate drawer holds a 96 and 384 well microplate or reservoir trough The instrument can simultaneously transfer a column of either Eight fluids from a 96 well compound plate to a 96 well assay plate or Sixteen fluids from a 384 well compound plate to a 384 well assay plate Important Be sure to install the compound baseplate before placing a compound plate in the drawer Reading Chamber Drawer Th...

Page 17: ... be opened by service trained users from the inside front panel if necessary for maintenance or from the inside top panel to install or remove the pipettor head The entire fluidics module can be removed for maintenance or to transport the system to another location For further information see 2 3 Installing the Fluidics Module See part 6 Troubleshooting Procedures for related service procedures In...

Page 18: ...with 96 well microplates In addition the FlexStation II includes a 16 channel pipettor head for use with 384 well microplates Note For further information see 2 4 Installing the Pipettor Head Eight or Sixteen Channel Pipettor Head Inside Top Panel Opened Pipettor Head 12 FlexStation II Operator s Manual Rev D ...

Page 19: ...sing to which the top cover is attached at the back of the instrument Reading Chamber Drawer with Assay Plate Top Cover Housing Reading Chamber Detection Module Fluidics Module Detection Module Detail Reading Chamber The reading chamber includes the assay plate carriage which holds the assay microplate in the reading chamber during read cycles The reading chamber may be maintained at an elevated t...

Page 20: ...ragile especially the excitation fiber Handle cables with extreme care Do not flex twist bend or stretch the optical cables Flash Lamp Photomultiplier Tube Excitation Monochromator Microplate Elliptical Focusing Mirrors Emission Monochromator Flash Lamp Collimation Mirror Optical System Overview of Optical System 1 The excitation light source is a xenon arc flash lamp Note that the lamp is off whe...

Page 21: ...OFTmax PRO you can choose which filter to use or use none or have a filter automatically selected 8 The PMT detects the emitted light and passes a quantitative signal to the instrument s electronics which then send the data to the computer 1 4 5 Computer The FlexStation II instrument works as a system with the SOFTmax PRO application software SOFTmax PRO must be installed on a dedicated personal c...

Page 22: ...shielded cables to connect the instrument to the computer Choose cables that meet the following requirements Serial Interface Cable IBM compatible Male DB8 to Female DB9 Note Contact Molecular Devices Technical Service for specific pin out requirements Microplate Adapters The black or purple microplate adapter fits in the assay plate carriage in the reading chamber drawer to elevate standard micro...

Page 23: ...oplates for bottom reading and all black microplates for top reading because they have lower backgrounds than clear plates For luminescence white microplates may optimize light collection Note Not all microplates are made with the same materials Some plastics most notably polystyrene have significant native fluorescence and can cause moderate to sever background fluorescence especially in the UV r...

Page 24: ...if needed Preparing and loading tips plates and compounds Preparing the software consists of Entering software preferences Selecting instrument settings Defining templates reduction parameters and display parameters Confirming hardware and software set up Running the experiment consists of Initiating the operation detection or fluidics plus detection Saving the data file Analyzing the data consist...

Page 25: ...vely small about 30 nm Exceptions to this rule include the lanthanide dyes used in time resolved fluorescence they have a Stokes shift that is typically about 300 nm There is considerable overlap between the excitation and emission spectra gray area when a small Stokes shift is present Excitation Maximum Emission Maximum Stokes Shift 1 0 0 5 0 500 550 600 650 Wavelength nm Relative Fluorescence Ex...

Page 26: ...o scan emission spectra is that you can assess more accurately whether the emission is due to the expected fluorophore multiple fluorophores or a variety of background sources or contaminants Another benefit is that if interfering fluorescent species are present you may be able to find excitation and emission wavelengths that avoid this interference For this reason it is desirable to scan emission...

Page 27: ...r from 1 C above ambient to 45 C Upon power up when the incubator is off the temperature in the reading chamber is ambient and isothermal Turning on the incubator by pressing the Incubator key will cause the instrument to begin warming the reading chamber and the fluidics module The temperature set point defaults to 37 C at start up With the incubator on the temperature of the reading chamber can ...

Page 28: ...er s manual Time Tagged Data Example Optical System The instrument uses excitation and emission filter wheels to decrease interference by stray light thus augmenting the wavelength selection that is provided by the monochromators Two independent single channel reading heads can service top and bottom reading requirements The top reading head supports coaxial excitation and emission beams in the ca...

Page 29: ...nd 384 well microplates Bottom reading functions in all detection modes Important Clear bottom plates must be used when using bottom reading Bottom reading is intended for cell based assays Note RFU scale is ten times 10 larger during bottom reading FlexStation II Operator s Manual Rev D 23 ...

Page 30: ...ne to 16 wells in one column of the assay plate are read repeatedly for a selected total experimental time At a preselected point or points during that time sequence the pipettor may transfer up to three reagents from the compound plate to the assay plate The instrument continues to read at the preselected time intervals before and after fluid transfer After completion of reading the column or par...

Page 31: ...No No Yes AutoRead Yes Yes Yes Yes Yes Well Scan Editor No No No Yes No PMT sensitivity Yes Yes Yes Yes Yes Assay plate type Yes Yes Yes Yes Yes Operation Modes and Features Note The system performs limited luminescence and TRF readings Endpoint Mode In Endpoint mode as well as in Kinetic and Flex modes you may select from one to four excitation emission pairs to obtain Relative Fluorescence Units...

Page 32: ...um mode the fluidics module is not enabled For more information on this mode please review the appropriate section in the SOFTmax PRO User s Manual Well Scan Mode Some applications that involve the detection of whole cells in large area tissue culture plates may require the use of Well Scan mode As many cell lines tend to grow in aggregates or in the edges of microplate wells this non confluent gr...

Page 33: ...e software uses standard Windows conventions for menus dialog boxes windows and mouse control These instructions assume that you are familiar with the basic operation of your computer This FlexStation II Operator s Manual describes using the SOFTmax PRO application to run FlexStation II experiments There is limited information about using the software for the other read modes For further details a...

Page 34: ...ong the tool bars or by using the pull down menus You can use either your mouse or keystrokes to make selections Note For complete details about the SOFTmax PRO application and user interface refer to your SOFTmax PRO Operator s manual Refer specifically to the following chapters Chapter 6 for a discussion of Flex mode setup Chapter 8 for information about data analysis in Flex mode Chapter 10 Tut...

Page 35: ...systems have different icons This Icon Has the Following Function The Instrument Status icon provides visual confirmation that SOFTmax PRO is communicating with the instrument Double click this icon to display Preferences dialog box Displays the current temperature inside the instrument Click this icon to display the Preferences dialog box Click to begin reading It changes to Stop during a reading...

Page 36: ...n create or edit the template This is used to group table setup for defining areas of the assay plate Click the Reduction icon to configure settings for data analysis and graph reduction Click the Display icon to open the Display dialog box and change your display properties Click the Graph icon to enlarge sections of the display into graphic form Click the Mask icon to mask selected wells Click t...

Page 37: ...mputer 2 6 Connecting the 2 7 Installing the Drawer Adapters 2 8 Installing SOFTmax PRO 2 1 General Precautionary Information Warning Always make sure the power switch on the instrument is in the OFF position and remove the power cord from the back of the instrument prior to any installation or relocation of the system Warning Do not install or operate the system in an environment where potentiall...

Page 38: ...system pieces examine the packing list that accompanies the system to be sure all items are present Unpacking the Fluidics Module and Accessories 1 Remove the fluidics module from the protective foam Set in a safe place 2 Remove the box containing the pipettor head and the bags of accessories 3 Open the accessories bags remove cables and the Allen wrench You will need them later in the assembly pr...

Page 39: ...t weighs approximately 55 pounds and should be lifted with care It is recommended that two persons lift the instrument together taking proper precautions to avoid injury 7 Set shipping materials out of the way Removing Shipping Screws 1 There are two shipping screws holding down the cover of the instrument Locate these stainless steel screws under the front flange of the detection module actual C ...

Page 40: ...on Procedures 3 Open the top cover by depressing the latch in the handle and pivoting the cover up and back Top Cover Reading Chamber Detection Module Opening the Top Cover 34 FlexStation II Operator s Manual Rev D ...

Page 41: ...attached to the flange and two locating pins near the middle of the reading chamber Top Cover Read Chamber Detection Module Hinged Flange with Quarter Turn Fasteners 2 Electrical Connector Fluidics Module Handle Holes for Locating Pins Locating Pins Positioning the Fluidics Module 2 Lift fluidics module into position Warning You may need two people to continue with this procedure The fluidics modu...

Page 42: ...ECTOR CONNECT FLUIDICS HERE Note There are labels on both the fluidics connector Fluidics Connector and near the communication port Connect Fluidics Here that help identify these parts 4 Lower fluidics module into place Caution Be careful when lowering the fluidics module that you do not trap or compress any of the optical fibers coming up from the detection module a Ensure that all cables and wir...

Page 43: ...place 1 Remove pipettor head from its corrugated plastic carton 2 Open the inside top panel of the instrument Turn the quarter turn fastener on the inside top cover and unfold the cover off the fluidics module to the right Opening Inside Top Cover 3 Attach round connector a Hold the pipettor head in your left hand and the round black 14 pin connector in your right hand b Move the pipettor head int...

Page 44: ...r loops of white spiral cord onto the coil hook in the upper left corner of the top panel opening This secures the cable up out of the way of the pipettor head when it moves about in the fluidics module during operation Coil Hook Securing Spiral Cord to Hook 38 FlexStation II Operator s Manual Rev D ...

Page 45: ... plate up into place Red Knob Alignment Pins Positioning Pipettor Head c Screw down the red knob securing the pipettor head so that it hangs in place from the black bar Caution Ensure the red knob the pipettor retaining nut is tightened as firmly as possible The pipettor head is now installed 5 Close the fluidics module Fold the inside top panel back over the pipettor head and lock the quarter tur...

Page 46: ...tting Up the Computer Set up the computer and monitor according to the instructions that come in their packaging Place them close to the instrument on the bench The power cords for the computer and monitor are provided in the computer packaging Connect them to the computer hardware see next section but not to the power outlet at the wall Caution Do not attach the computer to a power outlet until t...

Page 47: ...s box 2 Insert the 8 pin DIN round end of the serial cable into the RS 232 serial port receptacle on the back panel of the instrument Attach the other end to the COM serial port on the back of the computer 3 Insert the female end of the power cord into the power receptacle at the rear of the instrument 4 Connect the instrument power cord a grounded power outlet of the appropriate voltage Molecular...

Page 48: ...ter into the drawer first While pushing against the back edge of the adapter lower the front of the adapter into the drawer Compound Baseplate Installation Place the metal compound baseplate into the bottom of the compound plate drawer 1 Turn on power to the instrument 2 Press the Compounds button on the front panel The compound plate drawer opens 3 Lower the baseplate into the compound drawer wit...

Page 49: ... 3 2 Starting Up the System 3 3 Setting the Temperature 3 4 Setting Up the Software 3 5 Configuring Instrument Settings in Flex Mode 3 6 Other Software Settings 3 7 Reading the Microplate 3 8 Shutdown 3 9 Other Read Modes 3 10 Optimizing Fluorescence Assays in Spectrum Mode Note Information in part 3 assumes the instrument and computer are installed and connected properly For further information s...

Page 50: ... take a while for the temperature to stabilize so do this before configuring other instrument parameters Note Incubator settings can also be set using SOFTmax PRO 3 Configure instrument settings using SOFTmax PRO Configure the read mode type of analysis template etc as desired Create sections Notes and Plates as needed in the Experiment Section of the Software window 4 Load prepared pipette tip ra...

Page 51: ... FLEXstation 100 240V 5A 50 60 Hz T6 3 AH s n FS 01001 C NRTL C LR88026 PATENTS PENDING US Parallel Port not currently in use RS 232 Serial Port Power Cord Receptacle RS 232 Serial Cable Power Cord Fuse Box Cover Power Switch Power Switch Location The instrument automatically performs diagnostic checks to ensure that it is functioning correctly All three drawers open and shut After about four minu...

Page 52: ...n to start the program 3 View Untitled SOFTmax PRO window Note If you get an error message while the software is starting up see part 6 Troubleshooting Procedures After the SOFTmax PRO screen appears an Untitled window appears SOFTmax PRO New Untitled Window The first window comes up in Flex mode with a default template selected 46 FlexStation II Operator s Manual Rev D ...

Page 53: ... 10 of your SOFTmax PRO User s Manual Users familiar with SOFTmax PRO If you are already familiar with SOFTmax PRO software you can close the Notes section and open the Plate section You are now ready to begin setting up your experiment protocols Untitled Window Plate Section of Default Template Note For instructions on adjusting software settings see section 3 4 Setting Up the Software FlexStatio...

Page 54: ...ure shown in the display by an increment of 0 1 C press and hold to scroll tip rack Open or close the tip rack drawer compounds Open or close the compound plate drawer reading chamber Open or close the reading chamber drawer Whether or not the drawer will remain open depends on the incubator setting If the incubator is off the drawer will remain open if the incubator is on the drawer will close af...

Page 55: ... When the incubator is off this upper number will be the ambient temperature The lower reading is the set point that is the temperature you desire for the current experiment and it is displayed when the incubator is enabled actual C set pt C Set Temp Temp on off 3 3 2 Setting the Temperature with the Control Panel To enable the incubator press the Temp on off key on the control panel The display w...

Page 56: ...indow appears Incubator Dialog Window You can leave the temperature setting at the default value or you can type a different value into the highlighted box Note The incubator setting is independent of the protocol being run Running an experiment does not automatically select the temperature set point After a reading the temperature set point range and average actual temperature are recorded in the...

Page 57: ...that the instrument is communicating properly with the computer as part of routine software setup a Select Preferences from the Edit menu The Preferences dialog box appears Preferences Dialog Window b Make sure that the serial port setting agrees with the actual port the computer cable RS 232 cable is connected to This is usually Com1 c The serial comm speed should be 9600 Baud Note Once you read ...

Page 58: ...t Setup from the Control drop down menu You can also click on the Setup button on the Plate Section tool bar to view the Instrument Settings screen Instrument Settings Screen Notice that the FLEX button on the right is highlighted Note You can select a different read mode from the Instrument Settings screen by clicking on one of the other four buttons at the top of the window The rest of these ins...

Page 59: ... New Plate from the Experiment menu in the Menu bar to create more Plate sections If you wish to create a plate with settings identical to a particular existing plate highlight that plate then select Duplicate from the Edit drop down menu Double click on the Plate word in the Plate Section tool bar to open a dialog box and name your Plate sections Note Refer to SOFTmax PRO user manual for details ...

Page 60: ...issing any settings However the Timing setting depends on several settings which appear after it in the list You will need to return to Timing after completing all settings to obtain the minimum read interval It is essential that settings for the fluidics operations are correct and correlate with one another In particular layouts and settings for Assay Plate Type Wells to Read Compound Source Comp...

Page 61: ...lter Settings Select an emission cutoff filter that will block as much of the residual excitation light as possible without unduly reducing the fluorescence signal The cutoff wavelength options are 420 435 475 495 515 530 550 570 590 610 630 665 or 695 nm You can also select None if you don t want any cutoff The cutoff value should be near the maximum emission wavelength preferably between the exc...

Page 62: ...cence Assays in Spectrum Mode Important If you select an emission wavelength that is close to two times the excitation wavelength if EM λ 2 EX λ always be sure to use an emission cutoff filter to ensure you are blocking monochromator second order light If you run an experiment like this without an emission filter you will always observe an emission peak at two times the excitation wavelength but i...

Page 63: ...s with very low signal will you get improved precision by increasing the number of reads per well Note Increasing the number of reads also increases the total read time Sensitivity Settings The photomultiplier tube PMT is a photon detector that detects light through the use of photoemission and successive instances of secondary emission to produce enough electrons to generate a strong signal PMT S...

Page 64: ...erval The acceptable run time range is from zero to a thousand 0 1000 seconds When you have entered the values you desire the total Number of Reads calculated by the instrument is displayed automatically Depending on what sensitivity you have selected in the previous setting box the Minimum Interval and the Minimum Run Time will automatically adjust The minimum interval is also dependent on the Co...

Page 65: ...he right to indicate how long shaking should last Automix time can last from 1 to 999 seconds Automix Settings 3 5 5 AutoCalibrate This checkbox allows you to disable or enable automatic calibration The default is enabled Turn autocalibration off to allow the instrument to begin or complete readings more quickly Important Allow the instrument to perform an autocalibration of at least one plate bef...

Page 66: ...will be discarded The previously created groups remain however so you can select new wells and apply existing groups to them Note The Assay Plate setting takes precedence over all other fluidics module settings and will affect the correctness of other settings Important If you go back and change this setting after you have selected settings that follow for example Pipette Tips Layout Compound Sour...

Page 67: ...the entire plate Wells to Read Window Highlight the wells you want read with the cursor Wells must be contiguous and in a rectangular arrangement but do not need to start in the 1 column You may choose a partial row or column or a single well Note In planning your experiment remember that the instrument makes fluid transfers and readings one column at a time You may want to use partial rows A H ra...

Page 68: ... reading only those wells selected will be visible in the data display In the figure below the Plate section reflects that wells A1 through H2 have been selected for reading Plate Section with Selected Wells to Read 62 FlexStation II Operator s Manual Rev D ...

Page 69: ...ype setting In particular the well bottom height is different for different types of plates and selecting plate type correctly is important to prevent jamming pipette tips into the bottom of the well The instrument assumes a 20 µL pipette height when aspirating from a compound source plate See illustration below Compound Plate Aspirate Height 20 µL Default 10 µL for 384 wells Pipette Tip Well Bott...

Page 70: ...the Assay Plate Fluid box that equals the largest initial volume prior to compound transfers for any well in the assay plate This value can be set at 0 269 microliters µL although obviously typical values will be about 10 200 µL for a 96 well microplate SOFTmax PRO assumes all wells hold the same initial volume The software uses this value to compute the total volume in each well after all fluids ...

Page 71: ...is setting helps ensure that the tip of the pipette is below the surface of the liquid at the end of the transfer thus minimizing the possibility that undispensed drops remain on the tips Important As you configure subsequent transfers you must calculate the amount of fluid added and set the pipette height accordingly Rate 1 8 This setting determines the rate at which the fluid is dispensed into t...

Page 72: ... The calculation for the second event starts at the end of the first event and adds to that the total time that will be necessary to aspirate new fluids from the compound plate and dispense them into the assay plate Possible Problems Time point calculations are based on the number of wells read the tip column and compound column used during transfer If you select time points that are not long enou...

Page 73: ...rform trituration Then click in the box to select either Compound Source or Assay Plate or both You can set the Volume of fluid to be withdrawn from the well and the number of times Cycles it will be aspirated and dispensed back into that well In addition for assay plates you must also enter a value for the Height at which the trituration occurs The height setting should take into account the volu...

Page 74: ...o choose which tips are available in the Pipette Tips Layout setting do so with caution Be sure that the layout described in the software matches the actual tips inserted in the tip drawer Caution Molecular Devices recommends using a full rack of tips each time you perform a transfer of fluid in Flex mode If you mistakenly enable a pipetting function from a tip that is not present or if you enable...

Page 75: ...ated fluid will then be dispensed to the first available column indicated in the Wells to Read selection The software assumes All columns in the Wells to Read selection should receive fluid The fluids are transferred from left to right the read transfer read sequence in each column is initiated only after the previous column s read transfer read event is completed the total read time for that colu...

Page 76: ...ound column until all tips and columns have been targeted SOFTmax PRO does not fill beyond the limitations of the available Tips and Compound columns Once all available tips and compound columns have been assigned SOFTmax PRO simply stops assigning targets This setting window displays one two or three transfers with blue pink and green color coded tips to match the setting made in the Compound Tra...

Page 77: ...nd pop up menu The wells shown in this settings window convey graphically the volumes of liquid in the assay plate for each pipetting event Using the color associated with each event the compound settings display the dispensed compound volume as a percentage of the total assay well volume If you entered an initial volume in the Compound Transfer settings window that will be shown as a gray fill As...

Page 78: ...readings by typing a number in the Delay box Setting AutoRead Delay 3 5 14 Pipette Tip Air Gap To set the pipette tip air gap the volume between the end of the pipette tip and the bottom of the liquid in the tip select Select Air Gap from the Control menu to reveal the dialog box The value allowed is 0 200 µL for 96 wells and 0 30 µL for 384 wells Air Gap Pipette Tip Setting Pipette Tip Air Gap Pi...

Page 79: ...e figure below shows a Plate section with three sets of transfers The wells with blue squares in their upper left corners were selected for the first transfer wells with red triangles upper right corners were selected for the second transfer and wells with a green triangle lower left corner were selected for the third transfer Plate section with wells selected for injection The Plate section also ...

Page 80: ...lay parameters prior to reading the assay plate because these parameters determine how data is displayed and analyzed You can set up or modify templates reduction and display parameters after collection but this may be complicated or confusing Refer to your SOFTmax PRO User s Manual for information about using the software to continue to prepare for an experiment Note While it is strongly recommen...

Page 81: ...s by either manually pressing the appropriate drawer buttons on the control panel or by using SOFTmax PRO 2 Insert tips and plates Insert the filled tip rack and plates into the drawers placing well A1 into the upper left corner of the drawer as you look at it Make sure the compound plate is flat against the compound baseplate Make sure the assay plate is flat against the black adapter for 96 and ...

Page 82: ... If you have created more than one Plate section and no Plate section is active choosing the Read command will display a dialog box requiring you to choose which section to read Select the correct section and click OK If a Plate section is active starting a read will read the active plate Replacing Data in a Plate If you select a Plate section that already contains data and select the Read command...

Page 83: ...ays the amount of time elapsed since the beginning of the reading This box displays the portion of the reading currently under way Data Display You can observe the gradual accumulation of data in the Plate section of the SOFTmax PRO window The values read by the instrument will appear in the data display of the Plate section as they are received from the instrument to the software in real time See...

Page 84: ... laboratory s practice Note Normally you do not need to switch off the power at the end of the day If the system will not be used for more than one day it is best to turn off the instrument 3 9 Other Read Modes In addition to operating the FlexStation II instrument in Flex mode fluidics read the instrument can operate in four read only modes Endpoint Mode Kinetic Mode Spectrum Mode Well Scan Mode ...

Page 85: ...e for fluorescence the default is 6 for luminescence the default is 30 for time resolved fluorescence the default is 20 The voltage of the photomultiplier tube may be set to low for higher concentration samples medium or high for lower concentration samples in all read modes In Endpoint and Spectrum mode there is an additional setting automatic in which the instrument will automatically adjust the...

Page 86: ...an over a narrower wavelength range with a 1 or 2 nm increment e Perform scan and view the results as a plot of emission fluorescence vs excitation wavelength Note the excitation wavelength at the emission peak and the maximum RFU value Note If an error message reporting missing data points occurs it may be due to possible saturation reported by SOFTmax PRO at the end of the spectral scan Reset th...

Page 87: ...et up a second Plate section for a fluorescence read spectrum mode Ex Fixed Em Scan with no cutoff filter default and medium PMT b Set excitation wavelength to the value determined in 1f above c Set emission scan to start stop approximately 50 nm below above the tentative emission value obtained from the literature or existing filter pair d Set step increment to 1 2 nm or do a preliminary scan wit...

Page 88: ...the optimum filter as follows a First select an emission filter that is at least 20 nm larger than the selected excitation wavelength b Complete emission scan from the filter wavelength over a range of 50 nm to 100 nm larger c Compare signal to buffer background in the scan and choose the wavelength with the maximum ration d You may want to repeat the scan with the next larger emission filter and ...

Page 89: ...on wavelength Automatic Cutoff Selection Endpoint and Kinetic Modes Lambda λ nm Emission λ nm 1 None 415 2 420 415 434 3 435 435 454 4 455 455 474 5 475 475 494 6 495 495 514 7 515 515 529 8 530 530 549 9 550 550 569 10 570 570 589 11 590 590 609 12 610 610 629 13 630 630 664 14 665 665 694 15 695 695 850 Emission Cutoff Filter Default Settings Note For Spectrum mode the default is Manual that is ...

Page 90: ...kinetic fluorescence modes the Autofilter feature will generally select the same cutoff filter wavelength as will the above optimization method If desired however you may specify the cutoff filters manually b For emission wavelengths where there is no cutoff filter less than 420 nm or greater than 695 nm experimental iteration is usually the best method of determining the optimal emission and exci...

Page 91: ... reference Protocols Stay up to date with Molecular Devices FlexStation II Application Notes and Protocols You can locate these on the company s web site www moldev com Data Management See the SOFTmax PRO User s Manual for all information regarding data management FlexStation II Operator s Manual Rev D 85 ...

Page 92: ...4 Applications 86 FlexStation II Operator s Manual Rev D ...

Page 93: ...ion is a leading worldwide manufacturer and distributor of analytical instrumentation We are committed to the quality of our products and to fully supporting our customers with the highest possible level of technical service In order to fully benefit from our technical services please complete the registration warranty card and return it to the address printed on the card If you have any problems ...

Page 94: ...xStation II instrument in its original shipping cartons Otherwise carry the instrument or place it on a rolling cart to transport it 5 3 Cleaning the Instrument Biohazard Wear gloves during any cleaning procedure that could involve contact with either hazardous or biohazardous materials or fluids Periodically you should clean the outside surfaces of the FlexStation II instrument using a cloth or s...

Page 95: ...ter If you are reading high profile 6 12 24 or 48 well plates and the adapter is in the drawer you will need to remove the adapter 1 Turn power to the instrument on 2 Press the Reading Chamber button on the front panel or select Open Drawer from the Control menu 3 Hold the adapter at the front long side of the drawer and push toward the back Row A 4 Lift the front Row H of the adapter and remove i...

Page 96: ...r from a local hardware store Make sure fuses are rated slowblow 6 3 amp Use the following procedure for replacing the fuses 1 Disconnect power to the instrument a Turn off power to the instrument Remove the power cord from the outlet and from the instrument power cord receptacle b Remove the printer cable and computer cable if connected from the back of the instrument 2 Locate the fuse box a Turn...

Page 97: ...scard them c Insert new slow blow rated fuses into the fuse holder Either end of the fuse may be forward 5 Replace fuse box a Insert the fuse box into the opening in the instrument making sure that the fuses are on the right side toward the power receptacle b Press the fuse box into place making sure the cover snaps closed 6 Reconnect the instrument a Plug the power cord to the back into the instr...

Page 98: ...l WARRANTY MAY BE VOIDED unless lamp is replaced according to instructions in the operators manual under Flash Lamp Replacement Lamp Cover Flash Lamp Location Tools Required Allen and or ball driver hex wrenches 3 32 9 64 5 32 2 Phillips screwdriver 1 Turn off power Turn off the instrument power switch and unplug the power cord and the computer cable from the rear of the instrument 2 Remove lamp c...

Page 99: ...o vertical mounting cap screws that are holding the Litepac mounting block to the base plate They are located toward the inside of the lamp s mounting box The two cap screws will stay in a raised position spring loaded so that you can slide out the entire Litepac without the screws getting caught in the holes Note You may need a flashlight to see the cap screws Clamp Screw Flash Lamp Assembly Vert...

Page 100: ...ut the side of the instrument and lay it on its heat sink fins leaving the orange electrical cord connected Mounting Block Mounting Screws Spring loaded Flash Lamp Removing the Flash Lamp Assembly 6 Withdraw lamp from assembly a Use a 3 32 Allen wrench to remove the two screws that hold down the diamond shaped retainer Be careful not to lose screws Flashlamp Retainer Flashlamp Window Copper Coil S...

Page 101: ...ng block b Slide the lamp all the way into the block orienting the assembly to clear the base plate Slide it in until it stops c Tuck the orange power cord inside behind the assembly 9 Close the instrument top cover 10 Tighten the screws a Use the 5 32 hex ball driver to tighten the two vertical mounting screws which should still be captured Tighten them very firmly b Tighten the clamp screw snugl...

Page 102: ...5 Service and Maintenance Procedures 96 FlexStation II Operator s Manual Rev D ...

Page 103: ...e section 5 1 Technical Support 6 1 Problems During Startup Important Be sure the instrument is turned on before starting SOFTmax PRO After you start up SOFTmax PRO the Instrument Status bar should appear as in the illustration to the left with a temperature displaying in the temperature box Status Bar Detail If you see a red X in front of the instrument icon or if no temperature is displayed in t...

Page 104: ... the computer to which the instrument is physically connected It may be COM1 or COM2 b Make sure the Serial Comm Speed is correctly selected The communication speed is 9600 during the instrument s initial warm up sequence while it is first communicating with the software Note The Serial Comm Speed automatically changes to 57 600 when you select Flex mode from the Settings window If you follow thes...

Page 105: ...omputer cables from the computer Warning The power must be off for this procedure If the power is not disconnected and a jammed carriage or drawer is suddenly freed the instrument could suddenly move resulting in operator injury 2 Using your fingernail or a narrow blade inserted about inch open the drawer door to its horizontal position and pull out the carriage slowly and as gently as possible Ca...

Page 106: ...ation If the tips are accidentally released from the nose cones during operation they may fall outside of the rack If the power fails during a pipetting step serious damage can result to the instrument if appropriate corrective steps are not taken As can be seen there are several opportunities for mechanical problems within the fluidics module Fortunately these problems will be rare if you underst...

Page 107: ...ck plates and troughs if applicable securely in the correct drawers Make sure that the layout of the tips that you specify in your SOFTmax PRO protocol accurately represents what you have in the in the tip rack Make sure that the compound and assay plates that you specify in your SOFTmax PRO protocol accurately represent microplates you are actually using Note The software has no way of confirming...

Page 108: ...n include 6 4 1 Assessing a Mechanical Problem 6 4 2 Opening the Instrument 6 4 3 Evaluating the Tip Rack 6 4 4 Inspecting Inside the Fluidics Module 6 4 5 Removing the Pipettor Head 6 4 6 Expelling Undispensed Fluid from Tips Warning Always make sure the power switch on the instrument is in the OFF position and remove the power cord from the back of the instrument prior to removing any interior p...

Page 109: ...uidics operation follow the entire procedure until you can pinpoint the source of the problem Fluidics Error 1 Fluidics module not detected This message may occur if the fluidics module is not correctly installed Fluidics Error 2 Pipetter failure 1 This message may occur if the tip rack is jammed Tips may be missing Fluidics Error 3 Pipetter failure 2 This message occurs if fluid remains in the ti...

Page 110: ...le Detection Module Inside Top Panel Tip Rack Drawer Location of Inside Panels and Tip Rack Drawer 3 Remove front panel of fluidics module DO NOT REMOVE COVER UNTIL POWER IS DISCONNECTED FOR TRAINED SERVICE PERSONNEL ONLY Do not operate instrument unless all covers are in place Turn the four quarter turn screws and lift the front panel out You many need to inspect the interior of the module to det...

Page 111: ... out of the instrument to access the rack If all the tips are accounted for reposition the rack in the carriage Replace the inside front panel and top cover Reattach the power cord and computer cable to the instrument and reboot the instrument Save your SOFTmax PRO file to prevent loss of data then close and restart the software Important Failure to restart SOFTmax PRO may result in communication ...

Page 112: ... Caution Take care that you do not inadvertently dispel fluid before the pipettor head is outside the instrument Can you account for all tips or is there a possibility that some have dropped out of sight Do all the drawers move freely to gentle pressure or is there a plate or tip rack jamming them Once you have determined the status of pipettor head and tips there are a number of procedure you may...

Page 113: ... and dispel the liquid safely outside the instrument Caution Do not attempt to remove the pipettor head through the front inside panel See instructions in section 6 4 5 Removing the Pipettor Head If you are certain there is no liquid in the tips remove the tips from the pipettor head manually Reach into the fluidics module and gently pull the tips off the cones Gently push the tip rack and compoun...

Page 114: ...EX Icon No Communication If the red X remains after the warm up sequence select Preferences from the Edit menu of SOFTmax PRO and view the Preferences dialog box Preferences Dialog Box Make sure the Serial Comm Speed the communication rate or baud rate is set to 9600 Note When the FlexStation II instrument starts up it begins with a serial comm rate of 9600 Baud Upon initiation of a microplate rea...

Page 115: ...electrical connector lock and pull WHEN REINSTALLING INSURE PIPETTE RETAIN NUT IS FIRMLY TIGHTENED AS TIGHT AS POSSIBLE Biohazard Use proper handling precautions This procedure may include steps involving potentially infectious biological agents Follow your institution s protocol 1 Confirm that all power to the instrument is turned off 2 Unplug the power cord and the computer cable from the rear o...

Page 116: ...ad This will free the mechanism for easier removal Red Knob Alignment Pins Pipettor Head Detail Note If you want to dispense liquid from the tips skip the next two steps and see section 6 4 6 Expelling Undispensed Fluid from Tips next 8 To completely remove the pipettor head unscrew the black connector at the end of the white spiral cord from its secure position around the receptacle 9 Pull the co...

Page 117: ...oves the plungers and any liquid will be expelled and any tips will be ejected Replace the Pipettor Head Note See section 2 4 Installing the Pipettor Head for a more complete version of these next steps with illustrations 1 Slide the pipettor head back under its mounting plate inside the fluidics module making sure to secure the white spiral cord back on its retaining hook 2 Screw the red knob ret...

Page 118: ...plays the actual current temperature again However the red X is still covering the instrument icon on the SOFTmax PRO status bar 2 Select Recover from the Control menu In SOFTmax PRO select Recover from the Control menu The recover process is essentially a second startup cycle which restores the fluidics module to operating conditions and reboots the instrument Press Continue to recover the softwa...

Page 119: ...Mechanical Problem for error messages specific to fluidics operation failures General Error 1 No assay plate If you get this message check to be sure there is an assay plate correctly placed in the reading chamber drawer Start the reading again If you get any one of the following messages attempt your reading again before continuing with troubleshooting General Error 2 Unexpected response result c...

Page 120: ...t code General Error 7 Fatal error If you observe the following message try to determine if there are tips jamming the read head follow procedure in section 6 7 1 If this is not the case call Technical Support 114 FlexStation II Operator s Manual Rev D ...

Page 121: ...es if you have not been trained properly by appropriate Molecular Devices Personnel Warning Do not remove cover until power is disconnected Do not operate instrument unless all covers are in place Biohazard This procedure includes steps involving potentially infectious biological agents requiring that proper handling precautions be taken Follow your institution s biohazard protocol actual C set pt...

Page 122: ... Using the handle tilt the fluidics module to the left 4 Clean spill If you can clean up the spill or remove the tips without removing the fluidics module do so now If you need to remove the fluidics module continue with the instructions in the section below 5 Replace the fluidics module Using the handle lower the fluidics module carefully back over the detection module Make sure it is firmly seat...

Page 123: ...on chamber b Lift the fluidics module carefully off the detection chamber and set it on the bench 3 Clean up spill and or retrieve tips a Clean the spill If you are attempting to remove tips remove any obvious ones on top or around the sides of the reading chamber Look through the openings in the read head you may be able to see tips inside b If you see tips all the way down in the bottom tray of ...

Page 124: ...6 Troubleshooting Procedures 118 FlexStation II Operator s Manual Rev D ...

Page 125: ...ia 94089 Tel 408 747 1700 Fax 408 747 3601 US Canada Toll Free 800 635 5577 Online www moldev com Part Number Accessory 4400 0002 Power cord US 4400 0036 Power cord European 9000 0149 Computer cable 2300 0800 Top read microplate adapter reading chamber 0310 9336 Bottom read microplate adapter reading chamber 2500 1029 Compound Baseplate compound plate drawer 4600 0029 Fuse 0310 3992 Pipettor head ...

Page 126: ...ormats 6 12 24 48 96 384 Light Source Xenon flash lamp 1 joule flash user replaceable Lamp Life Two years normal operation one year warranty Detector Photomultiplier R 3896 Read Time 9 sec 96 wells 29 sec 384 wells measurement type may extend read time Single Column Read Frequency 96 wells 1 Hz Single Well Read Time Frequency 20 Hz Detection Spot Size Top Read 3 5 mm Detection Spot Size Bottom Rea...

Page 127: ...5 minutes for rated accuracy Operating Conditions 15 35 C Electrical Power Consumption 450 VA Line Voltage and Frequency 90 240 VAC 50 60 Hz Physical Size h w d 19 3 22 8 15 7 in 49 cm 58 cm 40 cm Weight 65 lb 29 5 kg FlexStation II Operator s Manual Rev D 121 ...

Page 128: ...ility Repair or replacement as provided under this warranty is the exclusive remedy to the purchaser the Buyer Molecular Devices Corporation the Seller shall not be liable for any incidental or consequential damages for breach of any express or implied warranty on this product except to the extent required by applicable law The Seller specifically excludes all express and implied warranties includ...

Page 129: ... 15 consumables 17 covers 7 8 detection module 13 14 drawers 9 10 fluidics module 11 12 optical system 14 15 panels 7 8 pipettor head 12 compound and tip columns 25 54 69 71 compound baseplate 16 installing 42 use of 89 compound plate 89 for each operational mode 25 loading 75 selecting 63 compound plate drawer 9 10 89 opening and closing 48 compound source 54 compound transfer 54 58 for each oper...

Page 130: ...communication speed 51 fluidics module 11 12 design 21 inspecting inside of 106 8 installing 35 36 tilting or removing 115 17 unpacking 32 fluorescence assays 79 84 fuses 16 replacing 90 91 G Graph icon 30 I icons Display 30 Graph 30 Instrument Status 29 51 Mask 30 Name of Plate 30 Plate 30 77 Printer 30 Reduction 30 Template 30 incubator 2 7 10 21 setting temperature 49 50 switching on 44 48 Incu...

Page 131: ...ates 75 77 setting the temperature 49 50 setting up the software 51 53 shutdown 78 starting up the system 45 48 optical system 14 15 22 overview of operation 18 44 overview of operator s manual 3 P panels 7 8 104 parallel port 8 parts replaceable 119 performance specifications 121 photomultiplier tube 57 sensitivity 25 setting voltage 79 pipette tip rack 16 17 loading 44 pipette tips 17 air gap 54...

Page 132: ...late 89 90 91 set point temperature indicator 48 settings instrument 54 73 assay plate 60 AutoCalibrate 59 Automix 59 AutoRead 72 compound and tip columns 69 71 compound source selection 63 compound transfer 64 66 finishing 54 modifying 54 on Plate Section screen 73 pipette tip air gap 72 pipette tips layout 68 sensitivity 57 timing 58 triturate selection 67 wavelengths 55 56 wells to read 61 62 S...

Page 133: ... 117 assessing mechanical problems 103 expelling fluid from tips 111 fluidics module inspecting inside 106 8 fluidics module tilting or removing 115 17 general error messages 113 14 mechanical problems Flex mode 102 12 opening drawers manually 99 opening the instrument 104 pipettor head removing 109 10 problems during startup 97 117 recovery procedure 112 tip rack evaluating problems with 105 unde...

Page 134: ...7 Appendices 128 FlexStation II Operator s Manual Rev D ...

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