Step 2: Add the same antibody or reagent used in samples. Use the selection guide to pick the correct compensation bead that binds the species or reagent and can be excited by the appropriate laser. Step 1: It is necessary for compensation beads to bind the antibody or reagent used in the specific experiment. Compensation beads are useful when they are as bright or brighter than samples used in a panel and when the fluorochrome spectrum are identical between sample and beads. Unstained samples and single-color controls are needed for setting parameters on spectral analyzers. All experiments must use single-color controls such as compensation beads to set gating parameters and optimize voltages for positive and negative signals.FMOs evaluate the effect of other fluorophores and compensation on background.Unstained sample can assess autofluorescence.Calculating compensation requires controls including unstained, fluorescence minus one (FMOs), and single-color samples: However, as the number of parameters and colors increase, so does the complexity of removing overlapping signal. Pairing fluorochromes based on antigen density, fluorochrome brightness, and separating by channels helps to minimize the effects from spillover and may remove the need for compensation from smaller experiments. Flow cytometry multicolor experiments may need compensation when there is fluorescence spillover ( Figure 1).
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