Most pre-conjugated antibodies contain sodium azide to minimise bacterial contamination. 20190122 - Do we need sodium azide free ABs for cell sorting? If the spillover is high then yes FMOs would be useful. There can be a use for these in determining gating positions, depending on the amount of fluorochrome spilling over into other channels. The 2 colour samples in this scenario could be called FMO controls (fluorescence minus one controls). I plan to have a no stain, and single stain controls. If licence is ordered at a later date it'll be charged pro-rata by month.Ģ0190304 - We are planning a three colour FACS Canto experiment. The FlowJo site licence billing cycle starts on the 1 st of March. 20190326 - Do you know when the next billing cycle is for the FlowJo site license option at Westmead Cytometry? FAM (essentially FITC) is excited well by the 488nm laser and again emits with a slightly longer wavelength that can be detected with the FITC detector on the CantoII. I’m wondering if it might be possible to do this on the CantoII? Or do I have to split my colours up into two experiments?ĪF647 is excited well by the red ~633nm laser and emits at a slightly longer wavelength. I have LDS-751 and EdU-click conjugation with AF647 and PNA-probe conjugated to FAM. To learn more about automatic compensation and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on Mastery Class wait list.20190517 - I am planning to do Flow-FISH using 3-colour system. Now if these three rules have been followed, and the compensation doesn’t look right, do not make the mistake of editing the compensation matrix to make the data look better.Īs many as 30% or researchers “tweak” the matrix to make it look better.įollow the above three rules and your compensation will be correct and if you have issues, explore what those problems are and work to resolve them rather than making up fiction by manual compensation. In every case where investigators have had “problems” with compensation, it is because they have violated the above three rules. For example, don’t use Alexa488 to compensate for FITC. The compensation color must be the same as the experimental color. Avoid using the universal negative for compensation.ģ. Background fluorescence should be the same between the negative and positive population. Controls must be at least as bright as the samples they will be applied to. Keys To Automatic Compensationįor automatic compensation to be successful, the following three rules must be followed.ġ. Manual compensation results in overcompensated data, yielding incorrect conclusions. The best practice is to use automatic compensation algorithms that are available in the most current versions of flow cytometry software that are based on the work by Bagwell and Adam (1993) Ann NY Acad Sci 20:167-184, who describe the mathematics behind multiparameter compensation. If you have manually compensated data in your lab notebook–strike it out now. Manual compensation is the process of adjusting the compensation based on how the data visually looks. One of the most common rumors or practices that has been passed down incorrectly by word of mouth over years past is the concept of manual compensation. Spillover is the overlap of a fluorochrome into a second channel due to the physics of fluorescence. This is a mathematical value that ensures that contributions from the fluorochromes not being displayed are not affecting the distribution of the data being displayed. There are a lot of rumors and mysteries that fill laboratory notebooks about the process. Some of these processes are correct while others lead to incorrect compensation, resulting in poor data.Ĭompensation is the process for correcting for the spillover. One of the most important steps in proper flow cytometry is the process of compensation. Written by Tim Bushnell, PhD / Figures courtesy of Pratip K.
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