What are MESF Units ?
Q. I've been working in a flow cytometry laboratory and I 'm wondering if there is any way to put a quantitative value on the fluorescence intensity of a cell?
A. Fluorescence intensity can be quantified by using standard units known as Molecules of Equivalent Soluble Fluorochrome (MESF).
Q. How are MESF units on the standards determined?
A. MESF units are determined by comparing the fluorescence intensity signal from the microbead standards to the signal from a solution of the same fluorochrome. Direct comparisons can be made in a spectrofluorometer between fluorochrome solutions and suspensions of microbead standards as long as the spectra match.
Q. Do MESF units tell you the actual number of fluorochrome molecules on the sample?
A. No. MESF units tell you how "bright" (intense) the signal is from the sample relative to a solution, not the actual number of fluorochrome molecules on the sample.
Q. What is the value in knowing the brightness of a sample in Molecules of Equivalent Soluble Fluorochrome ?
A. Equal numbers of fluorochrome molecules do not necessarily have the same brightness. Brightness needs to be corrected for changes in extinction coefficient, quenching, and small spectra shifts. MESF units account for most of these environmental corrections.
Q. What is quenching?
A. Quenching is the loss of fluorescence intensity due to energy transfer between molecules. It usually occurs when the molecules are closer together than 50 A.
Q. Is quenching the same as photobleaching?
A. No. Quenching is a reversible proximity, or concentration phenomenon, whereas photobleaching is an actual alteration or destruction of the fluorochrome by the excitation energy.
Q. Can MESF units be used with any fluorochrome?
A. Yes, provided that the specific fluorochrome is designated. For example, if fluorescein is used, then intensity is designated as MESF units of fluorescein. For Texas Red, the designation is MESF units of Texas Red.
Q. Most of my work involves antibody binding using two colors (FITC/PE). Do I need separate MESF standards for each channel?
A. Yes. It is important to match the excitation and emission spectra of the MESF standards with the FITC and the PE labeled antibodies. Again, you must designate the specific fluorochrome.
Q. How can I calibrate the fluorescence channels of my flow cytometer?
A. Calibration plots for each fluorescence channel can be made manually by plotting the peak channels of each of the different microbead populations against their MESF value (provided by the manufacturer). If you are using a log amplifier, be sure to make the plot using semi-log graph paper. If a linear amplifier is used, then log-log paper may be used to make the plot. Linear regressions of these parameters also produce the calibration and can yield a measure of linearity by means of the goodness of fit. It is important to use the right MESF standards with the right fluorescence detectorl, e.g., FITC standards with the FLl channel and PE standards with the FL2 channel. However, a program, QuickCal, is provided with each Quantum MESF Kit from FCSC which performs this linear regression.
Q. In what ways can I use the labeled quantitative microbead standards in my lab?
A. They can be used for day to day calibration of your instrumentation, to assure reproducibility in instrument performance. The standards can be used to give "dim" or "bright" antibodies a quantitative value (MESF). MESF intensities of cell populations can also be used as a quality control for staining or as an indicator of change in antibody binding capacity of the cells.
Q. How do I calculate the MESF values of an unknown?
A. There are two ways to solve for an unknown. The first is to use a calibration plot to determine the MESF value (y-axis) that corresponds to the peak channel of the unknown cells (x-axis). The other method is to use the regression equation and solve for MESF after entering in the channel value into the regression equation. Again, QuickCal will calculate the MESF value of unknowns when the peak channel is entered into the program.
Q. I have more than one instrument. Is there a way to correlate the values of samples between instruments?
A. Yes. By using MESF standards, you are able to compare the results of an unknown sample on multiple instruments by determining the intensity of the unknown in MESF units.
Q. How do MESF units relate to the number of antibodies binding to a cell?
A. There is a direct relationship between the MESF value of a cell population and the number of binding antibodies. The number of binding antibodies can be determined by dividing the MESF value per antibody into the MESF value of the cells.
Q. How do you determine the MESF value per antibody?
A. This can be determined by using a microbead that has a calibrated number of binding sites for the antibody and finding its MESF value when it is saturated with the antibody, then dividing its MESF value by the number of binding sites on the microbead. The MESF value per antibody is also referred to as the "effective fluorescence to protein (F/P) ratio".
Q. Are there MESF standards available to calibrate my flow cytometer?
A. Yes. Flow Cytometry Standards Corporation (FCSC) produces kits of fluorochrome-labeled microbeads known as the Quantum MESF Kits (FITC or PE). FCSC's Simply Cellular¨ microbeads bind calibrated amounts of antibody.
Q. Can I use QuickCal with any flow cytometer?
A. QuickCal¨ can be used with all flow cytometers, Becton Dickinson FACScan, FACStar, FACS Calibur and FACS Vantage; the Coulter Profile I, Profile II, EPICS C, Epics Elite and Epics XL; and the Ortho Cytoron.
Q. How do I get more information about these quantitative products from FCSC?
A. If you wish to obtain more information, order a catalog or place an order, please contact us at 1- (800) 227-8143 or write to us at Flow Cytometry Standards Corporation, PO BOX 194344, San Juan, Puerto Rico 00919-4344.
