Whole cell fluorescence reading Ex485nm/Em530nm with plate reader (SOP ID: 0004)
Last release date:
This Standard Operating Procedure provides a step-by-step protocol for whole-cell fluorescence reading using a plate reader with Ex485nm/Em530nm. This protocol includes 3 calibration sets (OD600 calibration, Particle standard curve, and Fluorescein standard curve) to ensure that collected data can be exchanged and compared among different laboratories using different equipment at different times. The rationale behind this protocol is to use reference materials and calibration curves to rescale readouts in order to make them directly comparable across different operators/labs/equipment. In particular:
LUDOX CL-X (45% colloidal silica suspension) will be used as a single point reference to obtain a conversion factor to transform absorbance data (Abs 600 nm) from your plate reader into a comparable OD600 measurement as it would be obtained in cuvette-based spectrophotometer. Absorbance conversion is required to account for well-to-well volume differences that may occur during plate set up. Any volume difference will affect the path length of the light passing through the sample in the well and hence affect the absorbance measurements. Although most plate readers are equipped with automatic pathlength correction feature, this is performed digitally after data acquisition compromising data reproducibility and comparability among different equipment. For this reason, this SOP requires the pathlength correction to be switched off and be replaced with a single point reference measurement as described in section 7.
Particle standard curve.
A serial dilution of monodisperse silica microspheres with size and optical characteristics similar to cells will be used as a standard curve to measure Abs600 and estimate the number of cells in your well.
Fluorescein standard curve.
Plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument. Therefore, absolute fluorescence values cannot be directly compared from one instrument to another. In addition, fluorescence readouts may be affected by process variables such as temperature and humidity. In order to compare the fluorescence output of different devices at different times, it is necessary to rescale readouts measurements to a standard fluorescence curve. This procedure uses the small molecule fluorescein, which has similar excitation and emission properties to GFP, as a surrogate of the GFP protein.
A serial dilution of fluorescein will be used as a standard curve to measure fluorescence with excitation at 485 nm (recommended) and emission 520-530 nm (recommended) with a bandpass width 25-30nm (recommended).
The figure below shows the typical arrangement of a microplate with the 3 standards. Wells A4 to H12 are then used for sample measurements.
- Standard plasticware
- fluorescence-compatible 96-well plate
4. Changes since last revision
5. Role and responsibilities
Author (Au): Staff scientist properly trained to draft, review and disseminate standard operating procedures within the framework of ISO-9001
Approver (Ap): PI with the authority to approve, reject and withdraw standard operating procedures
MIAMI Quality Manager (QM): MIAMI authorized Quality Manager
Operator (Op): Staff scientist properly trained to execute the standard operating procedure
Other (Oth): Third-party personnel and/or institution and/or authority, and/or service provider.