![]() The isoclonic control shows whether a fluorophore or other antibody conjugate is binding non-specifically to cellular components.Ĭells are stained with the conjugated antibody in the presence of an excess of identical (isoclonic) unlabeled antibody. To set up a secondary antibody control, we can use cells that have only been treated with the secondary antibody and haven't been exposed to the primary antibody. This type of control is essential when using a secondary antibody, as it will allow us to assess the extent of non-specific binding associated with the secondary antibody. Refer to our complete guide to isotype controls for more information. Be derived by the same manufacturing process and presented in the same formulation.Match the primary antibody in host species, class and subclass of heavy and light chains, fluorophore type, and number of fluorophore molecules per immunoglobulin.They should not be used to distinguish positive from negative cells or set positive gating regions. Isotype controls determine the level of background fluorescence caused by non-specific antibody binding. ![]() ![]() Isotype control is an antibody raised against an antigen not present on or in the cell type being analyzed. However, positive control cells might not always be available. This control allows us to avoid false negatives resulting from a faulty antibody. The positive control is the cells known to express your target of interest. ![]() Use this control to set gating regions and discern positive from negative cells. This sample should be exposed to the same experimental conditions as the population in the study. The negative control should be a population of cells that do not express the antigen of interest, ideally a knock-out cell line. Fc blocking to reduce non-specific antibody binding in flow cytometry. For example, in a multicolor panel of FITC, PE-Cy5, PE-Cy7, and PE, the PE FMO control would contain the FITC, Cy-PE, and Cy7-PE reagents but not the PE (Figure 2). FMO controlsįMO controls, samples stained with all antibodies in a panel except for one, are essential for providing a measure of spillover in a given channel and accurately discriminating positive and negative cell populations 1. This control provides a true negative control as it considers how the other fluorophores in your panel affect the signal observed in the channel used for the examined fluorophore. However, this can be controlled with compensation, where spectral overlap is estimated and subtracted from the total detected signal to yield an estimate of the actual amount of each dye, or including fluorescence minus one (FMO) controls to define the positive/negative populations. This phenomenon can result in false positives or incorrect gating when positive or negative boundaries are ambiguous. When carrying out a multicolor flow cytometry experiment, the emission spectra of the various fluorophores can overlap, resulting in detection in a different channel (also called spillover). Refer to our fluorochrome chart or multi-color selector to explore alternative lasers using your dye of choice. If there is significant autofluorescence, using a different laser wavelength can resolve the problem. To check if autofluorescence presents a problem in your experiment, analyze an aliquot of unstained cells on the flow cytometer using the same cell treatment and machine settings as the experimental sample. Naturally occurring cell components, such as NADPH and flavins, can emit fluorescence upon 488 nm wavelength laser excitation. Fixable Cell Viability Assay Kit (Fluorometric - Blue Ex 405 nm)įixable Cell Viability Assay Kit (Fluorometric - Deep Red)įixable Cell Viability Assay Kit (Fluorometric - Green)įixable Cell Viability Assay Kit (Fluorometric - Blue)įixable Cell Viability Assay Kit (Fluorometric - Green Ex 405 nm)įixable Cell Viability Assay Kit (Fluorometric - Orange Ex 405 nm)įixable Cell Viability Assay Kit (Fluorometric - Orange)įixable Cell Viability Assay Kit (Fluorometric - Red)Īutofluorescence is influenced by cell type and physiological conditions.
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