How To Choose Isotype Control For Flow Cytometry

You may choose not to use isotype controls but if you do there are some simple rules to follow which will ensure you use them properly and in conjunction with other controls.

How to choose isotype control for flow cytometry. Use the same isotype and subclass. Confirm the specificity of primary antibody binding. The level of fluorescence resulting from staining with the isotype control will be the non specific binding or the background fluorescence. Isotype controls should be used to determine the background due to nonspecific antibody binding.

The purpose of such a control is to. Abcam offers a complete range of isotype controls which are available both unconjugated and conjugated to. An isotype control will. Use the same isotype and subclass.

Use an isotype control that originates from the same host species as the primary antibody. Isotype controls are used to determine the background caused by nonspecific antibody binding. Rule out non specific fc receptor binding or other cellular protein interactions. How to choose an isotype control as a general rule of thumb try to match the following properties with the primary antibody.

Alexa fluor dyes hrp biotin phycoerythrin fitc agarose and other. Ensure the observed staining is due to specific binding rather than an artifact. Choosing your isotype control. Determine the non specific binding of an antibody to fc receptors found on monocytes macrophages dendritic and b cells.

Use an isotype control that originates from the same host species as the primary antibody. How to choose an isotype control as a general rule of thumb try to match the following properties with the primary antibody. F p is a measurement of how many fluorescent molecules are present on each antibody. In order to be useful the isotype control should ideally be the same isotype both in terms of species heavy chain iga igg igd ige or igm and light chain kappa or lambda class the same fluorochrome pe apc etc and have the same f p ratio.

Flow cytometry isotype controls the use of isotype controls in flow cytometry is controversial and divides researchers herzenberg l et al. To choose the proper isotype control for your experiment match the following characteristics of the primary antibody. Maecker ht and trotter j. Instead nonspecific staining should be alleviated by use of a blocking reagent the only valid use of an isotype control is to demonstrate that the samples were blocked sufficiently.

Reveal other non specific binding of the antibody or fluorophores to. An isotype control uses an antibody of the same isotype as the primary antibody but is specific for an antigen absent from the cells under study.