![]() ![]() The idea behind the Universal Negative is that a single tube, typically unstained cells, is used to set the negative population for establishing the compensation matrix. This blog article will help shine light on some of these historical practices and why they need to be changed. The last 2 blog articles have discussed the theory and practice of compensation. It is for this reason that there are suboptimal practices that permeate flow cytometry experiments to this day. The “truths” in this book are not always right anymore, but the new user doesn’t necessarily know any differently. Unfortunately, these pages are not refreshed with the best practices that have evolved over time as the technology and our understanding has changed and grown. These protocols are time-honored and tested, so the new researcher doesn’t question the wisdom of the “Protocols Book”. These hallowed, often coffee-stained, pages teach the researchers everything - from how to make media, passage cells, and run restriction digestions, to how to prepare cells for flow cytometry analysis. Employing fluorochrome combinations that have low cosine similarity scores (closer to 0) will generally produce better results with lower compensation values and lower spillover spreading, yielding increased sensitivity to detect low expressing antigens within populations that co-express that pair of fluorochromes.In most research labs, there exists a notebook that contains the tried and true protocols for lab members to follow. High cosine similarity scores (closer to 1) between fluorochrome pairs generally indicate higher compensation values and increased spillover spreading will occur between those two fluorochromes if used in the same staining panel, and these combinations may want to be avoided. This information can be used in the panel design process to inform on the degree of similarity between fluorochromes. In the context of flow cytometry, a CSM can be calculated from single color compensation controls, providing a pairwise comparison of the similarity between emission spectra of all fluorochromes employed in a staining panel. ![]() When calculating the cosine similarity of two fluorochromes, the component values of the vectors cannot be negative, in which case the cosine similarity is bounded in . ![]() The cosine similarity always belongs to the interval . For example, two proportional vectors have a cosine similarity of 1, two orthogonal vectors have a similarity of 0, and two opposite vectors have a similarity of -1. It follows that the cosine similarity does not depend on the magnitudes of the vectors, but only on their angle. That is, it is the dot product of the vectors divided by the product of their lengths. Cosine similarity is the cosine of the angle between the vectors. The CSM can be visualized and/or exported from the Compensation Matrix Preview/Edit window as shown below.Ĭosine similarity is a measure of similarity between two non-zero vectors defined in an inner product space. In FlowJo 10.9.0 and later, a cosine similarity matrix (CSM) is automatically created when a compensation matrix is calculated in the compensation wizard.
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