Sorry I meant to say, the macrophages are derived from human monocytes from whole blood.

Hey Gabriel,

I suspect there would be a degree of non-specific binding in the absence of Fc block. You have a few options for negative staining controls in this case:

1. isotype, as you mentioned

2. FMO (where you include everything in your master mix other than the antibody of interest)

3. Biological negative - if you have a cell type that you know doesn't express these markers this could be an option.

Also, whether or not people need an negative staining controls depends greatly on the panel and the antigens of interest. It is very common for people to use negative controls for antigens with continuous, instead of discrete expression. For example, you may want to use a negative staining control for cytokines, and co-stimulatory molecules, but not for CD45.

Hi Gabriel,

Completely agree with WH Tomaszewski's comments.

In addition, it is important that you Fc-block your samples. You expressed some concern about blocking and staining for CD16, but if your samples are human you can use anti CD16 clone 3G8, which is compatible with Fc-blocking.

Also, it's been shown that monocytes and macrophages can bind tandem-dye fluorochromes in an unspecific manner. There are reagents (check the True Stain Monocyte blocker from Biolegend) that can decrease this unspecific binding. using this reagent, combined with Fc blocking will give you cleaner results.

About using isotype controls, here there is a link with useful comments that might help you to decide whether or not to use them in your experiment.

https://expertcytometry.com/when-to-use-and-not-use-flow-cytometry-isotype-controls/

Hope this is useful.

Regards

Thanks ever so much Fabian Flores-Borja and William H Tomaszewski for your responses. That's interesting that 3G8 is compatible with an Fc block. That is the clone I am currently using. Do you refer to recombinant Fc blocks such as BD's https://www.bdbiosciences.com/documents/Fc-Block-DS.pdf , as opposed to human serum?

Also that's very interesting with regards to the unspecific tandem dye staining - that's not something I had previously heard of. I am currently using Pe-Cy7 so will definitely look into purchasing that - thanks for the information.

Hi Gabriel,

I'm not sure what the composition of the BD Fc receptor-blocking reagent is, but human serum or purified gamma globulins are fine for Fc receptor blocking in human cells.

PE-Cy7 is one of the fluorochromes that monocytes-macrophages can bin in non-specific manner.

This is the reagent from Biolegend, here it is mentioned which other fluorochromes show this non-specific binding:

https://www.biolegend.com/en-us/products/true-stain-monocyte-blocker-14598

Regards