Plate reader instructions

Plate reader instructions

Computer

The computer controlling the plate reader runs Windows XP. Some familiarity with Windows should be sufficient to use it.

Accounts on computer

  • One for each lab, usually named after PI
  • User can change password
  • User can create password reset disk in case password is lost
  • Quota for storage space-files small so no-one has exceeded quota
  • Account is limited user type so you cannot install programs
  • Welcome picture, wall paper, quick launch toolbar items can be changed by user
  • Computer not connected to Internet to simplify computer management.

To log on, either scroll through list of accounts on Welcome screen, or press Ctrl-Alt-Del twice to bring up a log on box (faster).

Data can be moved from the computer by writing it on a flash drive (; ports are on the left side of the monitor and on front of computer, under cover below power button), CD or a diskette.

Start SoftMax Pro using the icon on the Quick Launch toolbar or on the desktop.

There should be an icon for the manual on the desktop. Your files should be automatically saved to the My Documents folder with the autosave feature. In addition, when you manually save data, (File-save as) it should be in the My Documents folder until you change it.

After starting the program, you should find a list of protocols on the Assay menu. The list is a standard grouping supplied by Molecular Devices and includes some which cannot be used on this instrument; they can be deleted. You can add your own protocols.

You can save new protocols. Protocols from other labs will be inaccessible; this is to reduce the size of the protocols list. When you save a data or protocol file, the folder used will be the last folder used, which normally should be your My Documents folder. The program as delivered used the \Program files\Softmax Pro folder. You can read files from that folder, but not store files there.

If you find the computer screen is turned on (green light on power button) but blank because the screen saver has activated, move the mouse to bring the display up. You will have to log on to your account. If SoftMax Pro does not start, it is probably because someone else left it running. The simplest solution is to restart the computer (Start button, Turn off computer then restart. Ignore the messages about others risking losing data and say yes to restart.) After using the plate reader, close SoftMax Pro, and turn off the plate reader. Turn off the computer and display.

If the computer does something strange so that restarting it seems the appropriate response, press power button and keep it depressed until the computer switches off; this will take several seconds.

Gemini EM plate reader

Capabilities

The instrument is a dual monochromator fluorescence instrument. This design enables it to read a wide range of fluorescent compounds. It cannot read absorbance.

Specifications

Wavelength ranges: 250 nm to 850 nm, excitation and emission, 9 nm bandwidth.

Sensitivity (top read, signal 3X standard deviation of baseline) 3 fmole FITC in a 200 µL well; bottom read, 8 fmole of FITC

No capability for polarized fluorescence

Time resolved fluorescence- a secondary mode

Data collection: 50-1450 μsec., 200 μsec increments

Sensitivity: 0.5 fmol/well Eu-chelate (obtained with DELFIA® reagent from Perkin Elmer)

Luminescence a secondary mode

Detection limit: 10 amol/well alkaline phosphatase 200 μL/well (obtained with Emerald IITM reagent from Applied Biosystems).

Because there are no injectors, it cannot perform flash luminescence like the Promega Dual Glo Luciferase assay.

The limited control over read time limits sensitivity.

Note that plates exposed to light may emit light for some time, increasing background.

Plate types
Strips of wells should be avoided because they carry a risk of the strip falling into the machine requiring an expensive, time consuming repair by the manufacturer. If they must be used, rigid frames are recommended, and the strips must be securely installed.

Thin walled flexible plates also seem unsuitable because they may jump out of position when the drawer closes.

Suggested plates for best data:

Fluorescence-top read

Solid Black flat-bottom for Excitations above 350 nm

Solid white flat-bottom for Excitations using deep UV (250-350 nm)

If culture inspection is required, Solid Black with clear flat-bottom

Fluorescence-bottom read

Solid Black with clear flat bottom

Luminescence, Glow luminescence

Solid White; these plates may emit light after exposure to room lights. Observe background to see if they decrease with time.

Operation

The instrument takes several minutes to perform start up tests.

After the instrument is ready for use, and you have started SoftMax Pro, a window appears showing a plate and a setup icon. Suggestions and explanations for the options are listed in the SOFTmax Pro manual p. 269:

To set the instrument for your reading, you can look for an existing method under the Assays menu item, or enter setup  by pressing the setup button in the window, or double click the grey area with setting, or on the top menu go to Control-Instrument Setup

Type of data

Choices are;        endpoint-a single reading of each well
kinetic-timed readings
spectrum
well scan-multiple readings of one well


read type.
Fluorescence is the default. Top reading gives greater sensitivity for 96 well plates, unless you have cells adhering to the bottom of the well.(i.e. leave bottom read box blank)
The instrument has some capacity for luminescence readings, but does not have the capabilities of a dedicated luminometer. The instrument cannot perform a dual luciferase assay (e.g. Promega).
Time resolved fluorescence is for compounds which emit light after the lamp is off; this mode reduces background noise.

Wavelengths
Either select from drop down list or type in the wavelength you want for excitation and emission. The auto cutoff box sets filters; leaving it on is suggested except for scans. Note that more than one wavelength can be read.

Sensitivity
The photomultiplier tube setting can be changed for samples with high or low concentrations. The voltage on the tube is changed. This is equivalent to gain.

The number or readings can be increased to increase accuracy, at the expense of time. Maximum number of readings is 30.

automix,
This setting is to shake the plate. Leave off unless you know you need it.

autocalibrate;
This is an instrument calibration and should be done at least once per session. Omission of autocalibrations reduces time for readings.

Assay plate type
a list of plates specifically supported shows, 96, 384, 24, 12, 6 well types. Use standard plate unless you see your type listed. Suggested plates are black walled. Clear plates will give more cross talk between wells.

Wells to read
Dark blue indicates well will be read. Part of a plate can be selected so that only a selected area of the plate is read.

Auto read. Used if you define different plate sections; see p. 39 of SOFTmax PRO manual.

You can save your settings as a protocol under File-Save As. In the dialog box, for file type, choose Protocol (ppr). This protocol should now appear in a list under the Assays menu.

A saved assay protocol will put in settings for wavelength.

Other features

Temperature can be set to 4º above ambient to 45°C (thermometer icon) on main toolbar

The plate can be shaken (icon right of thermometer, and see automix option in setup).

Unless reading plates from the bottom, put plates on the purple adapter to put them closer to the reading head. Remove plate cover.

Press read button on menu on top of page.

Wavelength settings

The manual for the plate reader describes how to determine the optimal wavelength settings for a fluorophore. If excitation and emission maxima are less than 80 nm different, see the documents below on how to find the best wavelength settings.

Alexa dyes are one class of compound that need careful choice of wavelengths. See documents:
30-Wavelength Selection on Gemini
31-Wavelength Optimizing on Gemini
on the desktop of the user accounts.

For some common wavelengths, see

  • Spectra Max manual, appendix, page B-3
  • Molecular Probes website or catalog
  • Fluorophore list 2 on the desktop of the computer (a pdf file)

If you have two fluorophores, you may have better separation of the two signals by using non-peak wavelengths.

Problems

Instrument does not work.

A few times the instrument did not work, because the wrong instrument selected in software. After the instrument has completed startup checks, in the top left of the computer screen, just under File, there is an instrument icon. If there is a large red X after the instrument display says the instrument is ready, there is a problem.

Click on the icon to bring up a Preferences window. On the left side of the window, near the top there is a box for serial port. It should be COM1.
In the lower half of the window, left side, there is a box labeled reader. The entry should be SpectraMax GEMINI EM. If it is something different, click on the arrow to bring up a list of instruments, and select the correct entry.

This information seems to be saved in the folder for protocols, so will not be saved if the folders are stored in the read only folder Program Files\SoftMax Pro 4.7\.

Low sensitivity

Note that a versatile monochromator based instrument like the Gemini EM is less sensitive than one using filters; the advantage is that it is easy to set the Gemini for a wide range of compounds.

Standard things to check if the sensitivity is low:

Top read setting?

Use of purple plate adaptor for top read

Is there something colored in the sample which absorbs light?

Try setting PMT to high in assay method

Increase number of readings to 30 in assay method to reduce noise

Optimize wavelengths-protocols from Molecular Devices are on computer desktop

Use all black plate

Consider non binding surface type plate (Greiner and Corning are 2 manufacturers)

Note that the instrument self check should detect a weak lamp.

Setting standards, unknowns, dilutions

Click template button. A new window should appear. To designate wells as blank, standards or unknowns, click on template. Then select wells, and then click on the dropdown list next to group in the top left corner to select the well type. If the well type is something other than blank, click new.
In the group settings window, click on list under group format.
For unknowns, you can enter a dilution series.
For standards, select cells, click the list for group, select new, then in the group settings window, under column format, select standards. If you click the sample descriptor box, you can chose the name for the standards and units.

After drawing up a template, you can save it. One method is under the Plate menu at the top of the screen, chose Export template. To use the template, go to Plate-import template. Then select cells, and click the Group button, and select type of sample from the drop down list. The wells should then have a label. Note the series button, useful if you have standards that increase in a fixed step.

Data-saving and moving

There is an autosave feature with a default name. An addition, data can be manually saved so that you can give the file a name which helps identify the data.

Data can be saved in a format recognized by Excel;go to
File-export.
For file type, choose text.

The default is to export data in a plate format. Excel normally recognizes how to open the file.
To export in time format, go to Edit-preferences. In the left side of the window is a box for data export format, either time or plate. Time format is useful for kinetic data.

Data is saved in .pda files.

To get data from the computer, the choices are:

Printing

Saving to a floppy diskette

Writing to a CD

Writing to a USB flash drive.; ports are on  the left side of the monitor and on front of computer, under cover below power button.

Software-SoftMaxPro-data analysis

Many people export the data to a text file (File-export. File type text) and then analyze in Excel.

The SoftMax program can be installed on a limited number of other computers; the serial number of the software is needed, the instrument serial number is irrelevant.

Help

Near the instrument is a book from Molecular Probes with advice on reading fluorescent samples.
The software manual is also present, and on the computer desktop.

Life Science Technical Sales Representative

Molecular Devices Corporation

800.635.5577

MDC web site:     http://www.moldev.com/