Fluorescent Plate Reader

Fluorescent plate readerThe Biomolecular Analysis Facility has a Molecular Devices SPECTRAmax  Gemini EM fluorescent plate reader. The instrument is located in  Pinn Hall room 1074, in the Biomolecular Analysis Facility.  Users need to contact Dr. John Shannon of the Biomolecular Analysis Facility at 434.243.9399 or by email at jds1c@virginia.edu to establish an account on the computer which controls the instrument. Dr. Shannon will offer basic training for the operation of the instrument. Instructions to get started are available. Most users need only a short time to take readings, so it is unusual to experience a wait to use the instrument, hence reservations are not required for standard use.

Some applications for the instrument are

  • measuring live and dead cells in the same well, using Promega MultiTox reagents to measure proteinases specific for live and dead cells. This assay takes advantage of the instrument’s ability to measure two fluorophores in the same assay and to scan wells to look for clumps of cells.
  • measuring glycogen  content of cultured cells
  • quantitation of DNA and RNA with PicoGreen or other dyes
  • cathepsin assay
  • measuring relative cell numbers with Alamar Blue
  • intracellular calcium
  • sensitive protein estimation
  • cholesterol assay
  • one user is quantitating DNA at 5 ng/mL.

Features of the SPECTRAmax instrument are

  • dual monochromators (bandwidth 9 nm) allowing any fluorophore to be read within the range of 250 to 850 nm
  • fluorescent spectra capability
  • top or bottom reading of plates for adherent cells
  • well scanning
  • kinetic measurements
  • 384, 96, 48, 24, 12, 6 well plates
  • heating of plates
  • shaking of plate
  • SoftMax Pro 4.7 software for data analysis.
  • claimed sensitivity of 3 fmol/well of FITC with top reading

This instrument does not measure absorbance.

It can perform some time resolved fluorescence measurements but not polarized fluorescence.

The instrument can be used as a luminometer for some applications. It does not have the sensitivity of a dedicated luminometer, nor does it have injectors required for flash luminescence assays e.g. Promega Dual Glo Luciferase assays.
Detection limit: 10 amol/well alkaline phosphatase 200 μL/well (obtained with Emerald IITM reagent from Applied Biosystems).

The instrument is user operated. The current charges for use are $15 for the first 10 minutes, $15/hr after the first 10 minutes.

For top read fluorescence, all black plates are recommended For luminescence, white plates which should be kept out of light to reduce light emission from the plastic.