Let’s think about a simple problem that starts with an observation and apply the scientific method to solve the problem. What are the ethical implications of human cloning?.How effective is this drug in treating this disease?.Which is better: classical music or rock and roll?.Do birds prefer bird feeders of a specific color?.What is the optimum temperature for the growth of E.Questions that cannot be answered using science Questions that can be answered using science The scientific process typically starts with an observation (often a problem to be solved) that leads to a question. Remember that science is very good at answering questions having to do with observations about the natural world, but is very bad at answering questions having to do with morals, ethics, or personal opinions. Figure 1 Sir Francis Bacon (1561–1626) is credited with being the first to define the scientific method. The scientific method is not exclusively used by biologists but can be applied to almost anything as a logical problem solving method. The scientific process was used even in ancient times, but it was first documented by England’s Sir Francis Bacon (1561–1626) ( Figure 1), who set up inductive methods for scientific inquiry. This approach is common to other sciences as well and is often referred to as the scientific method. In order to live up to the Facility's mission of assuring quality control and reproducibility, facility staff will not assist with running samples when the necessary compensation controls are not provided by the investigator.Biologists study the living world by posing questions about it and seeking science-based responses. The lack of proper compensation controls may yield misleading, confusing, and inaccurate data. Please Note: The facility's mission is to serve investigators in their quest to obtain accurate data. * any antibody that is compatible with the beads See Tandem Fluorochrome Conjugated Antibody Best Practices There are exceptions to these rules when using tandem fluorochromes. tube 4) anti-CD8 APC stained splenocytes (or antibody against some other high density antigent).tube 3) anti-CD8 PerCP stained splenocytes (or antibody against some other high density antigent).tube 3) anti-CD8 PE stained splenocytes (or antibody against some other high density antigent).tube 2) anti-CD8 FITC stained splenocytes (or antibody against some other high density antigent).Because of the necessity to have brightly stained cells at a relatively high frequency (i.e, above 10% of the population) for accurate compensation, it may be necessary to use the same antibody that stains the high density antigen while varying the fluorchrome for each tube (see the following example), except when using tandem fluorochromes (see following section about using tandem fluorochromes).Įxample: For mouse splenocytes stained with FITC, PE, PerCP, and APC conjugated antibodies, compensation controls should include: If beads are not used, then cells expressing high levels of antigen (does not have to be an antigen of interest in the experiment) are stained with a fluorochrome-conjugated antibody that yields brightly stained cells. For experiments that cannot spare cells for compensation, do not have enough positive events, or have only low antigen expression, compensation beads are recommended. The beads are stained as if they were cells using the same antibodies and fluorochromes that are used in the experiment, producing both a negative and bright positive population for each color. Several vendors sell beads specifically for use as compensation controls. It is important that each compensation tube have a population of brightly stained cells (or beads) in order for the spill-over values to be accurately determined. Each of the compensation tubes is subsequently run to establish the spill-over values of each fluorochrome into the other fluorescent channels. The negative control (unstained cells) establishes the background fluorescence of the experimental samples and is used to set the baseline PMT (photomultiplier tube) voltages of the instrument. The proper compensation controls include a negative control (unstained cells are recommended) and one tube each of cells (or beads) stained positively with each of the fluorochromes used in the experiment. Compensation controls MUST match the exact experimental fluorochrome.Background fluorescence should be the same for the positive and negative control (e.g, positive cells vs negative cells, or positive beads vs negative beads).Controls need to be as bright or brighter than any sample the compensation will be applied to.(See for an in-depth explanation of compensation.) Spectral overlap between fluorochromes in multi-color experiments requires the use of fluorescence compensation controls.
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