Dear Han,

If you are using Isotype control, it is mendatory to use same isotype with same fluorochrome as of the antibody of interest.

Using isotype of different chain of antibody, is wastage of reagent and labour.

Attaching you the file for difference of different isotypes and their binding affinity.

Hope you find this helpful.

Best of luck

Hi Han Chiu,

The short answer is "NO".

In order for an isotype control to be relevant, you must match the following characteristics with your specific antibody: Isotype, Species, Fluorochrome, F:P ratio, and Concentration.

Even after you make sure all of the above characteristics match, differences in the amino acid sequence in the variable regions (of Ab vs Isotype) have been shown to cause differential non-desirable adherence.

For this reason, people have stopped using Isotype controls altogether (especially, where the distribution is bimodal and is very easy to gate around positive populations by simply using an unstained control).

For long answer: here is a nice article written by Tim Bushnell from Excyte: https://expert.cheekyscientist.com/when-to-use-and-not-use-flow-cytometry-isotype-controls/

Of course, for an isotype control you would need the same isotype. If you don't have a product marketed as an isotype control, you can use any unrelated (known not to bind your cells specifically) antibody (same isotype/fluor). For instance, you can use; an anti-CD3 antibody like an isotype control for B cells, or you can use an intracellular antigen for surface staining, wrong species, etc.

Best answer, the company technical support will provide for primary, they know exact what works.