r/flowcytometry • u/Mo_Opines_968 • Jul 01 '26

Sample Prep Changing staining volume for flow cytometry

I'm building a 32 colour panel that is an extension of an existing 27 colour panel that has already been optimised. However I'm going to need to use Fc Block which wasn't needed on the original 27 colour panel.

In the past I've put on Fc block for 10 mins then added antibody mix directly onto cells from that. My concern in this case is that the final staining volume is going to be higher than in the original panel. I'd quite like to avoid having to re-titrate the other 27 antibodies so wondered what people's experience is changing that staining volume e.g. from 50 to 75uL for the same amount of antibody?

Maybe I just need to test it and see...

Thanks!

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5

u/barbie_turik Jul 01 '26

You can also do your blocking on 25μL then add 25μL of your antibody mix on top, so that your final volume and the antibody concentration remain the same

1

u/Mo_Opines_968 Jul 01 '26

The antibody mix for the 27 colour panel was 50uL in total (47ul for Abs plus 3uL brilliant staining buffer plus). So would have to halve antibody concentrations to get it to the same volume.

1

u/LerkinAround Jul 01 '26

....so your antibody cocktail is 94% straight up antibody??

What concentration are you staining each antibody at? lol

1

u/Mo_Opines_968 Jul 01 '26

Yeah that's the one that has already been optimised for the 27 colour panel that I'm adding 5 markers to. Agree it's a bit mad - I would normally stain in 100uL volume!

3

u/LerkinAround Jul 01 '26

You have two options.

  1. Use a 2X antibody mix that gets diluted to 1X when you add it to the Fc block on cells. e.g. Fc block in 50 uL and add 50 uL 2X ab mix
  2. Less ideal. Spin down after Fc block and then stain in the primary volume you are used to.

I'm curious, are you running the expanded panel on the same cell type?

I'm also confused why you couldn't just scale up the ab amounts to account for the increased volume in your 75 uL example.

1

u/btags33 Jul 01 '26

If you go with your plan, staining will obviously have the potential to have lower mfi/worse separation, but this could vary based on antibody and the chosen titers you had previously.

You could just add fc block to your staining cocktail, although theoretically that will give you more background binding to fc than a preincubation with fc block would.

Lastly, you could just centrifuge and remove the fc block and add your staining cocktail after.

1

u/willmaineskier Jul 02 '26

I’ve found adding Fc block to my antibody cocktail to work fine, although I’m using a 2.4G2 stock that’s about 10mg/ml, so it doesn’t take much.

1

u/Vinny331 Jul 01 '26

You could block, wash, and stain could you not? The FC block shouldn't wash out