![]() This type of binding can lead to false positives, reduced resolution between the positive and negative cells, and poor data (Figure 2). ![]() They bind antibodies via the constant Fc domain, rather than the antigen-specific Fab domain, leading to multiple antibodies binding to unintended targets. Red, lymphocytes blue, monocytes green, granulocytes.įc receptors are found on monocytes, macrophages, dendritic cells, and B cells. The autofluorescence of different populations can be clearly observed in the histograms. Unstained peripheral blood.Unstained peripheral blood was used as a negative control to set the FSC, SSC, and PMT voltage (561 nm laser, 577/15 filter shown). However, unstained cells should still be acquired to determine the level of autofluoresence, which may be different in a heterogeneous population such as peripheral blood for the different cell types (Figure 1).įig. For this method, select the voltage that gives the maximum difference between the first and second fluorescence peaks to ensure optimum PMT sensitivity. Then make minor adjustments if necessary to avoid very bright cells from being off the scale. Minor changes may still be required, but you will minimize the amount of precious sample and time required to set up the experiment.Īn alternative method for setting PMT voltages uses beads dyed with eight different fluorescence intensities. You will be able to save these settings ready to be uploaded in preparation for future experiments. This will allow you to determine the level of background fluorescence or autofluorescence in your sample (Figure 1) and set your voltages appropriately for each fluorescence channel, ensuring all signals can be detected. Then set photomultiplier tube (PMT) voltages so that negative cells and dim signals can be distinguished from electronic noise while keeping bright cells within the scale. Use unstained cells to set up the instrument so that all of your cells can be easily visualized on forward scatter (FSC) and side scatter (SSC) plots. This will help you choose compatible fluorophores which can be easily detected and pick the optimal filter set. Here you will learn about the essential controls you should include in your experiment and when to use them to obtain publication quality data.īefore starting an experiment, familiarize yourself with the instrument you will use so you know which lasers and filters are available. ![]() Controls are vital to any flow experiment to reliably distinguish your results from background variation and nonspecific effects.
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