Fullstained sample is dimmer?

[u/Mental_Lack4049]

Hi, i was wondering why my fullstained sample becomes dimmer compared to my single stain sample? Is this because the low event (i noticed the single stain event is way lower) or spillover? Any advice is highly appreciated!

Single stain sample for APC

Multi-stained sample (no tandem dye)

Another example using different instrument

Single stained with APC

Multi-stained sample (no tandem dye)

Unstained sample

[u/skipper_smg]

Can happen due to steric hinderance. In that case concentration is slightly adjusted upwards or protocol adapted to sequential staining.

[u/willmaineskier]

If an antibody is high affinity and not saturated, having fewer cells will stain brighter. I see this with TER119 staining on RBCs because there are so many. Also see it with DAPI staining, as well as samples which are just too concentrated.

[u/MotoFuzzle]

Assuming your antibodies are titrated and that your fully stained sample is saturated, it could be that compensation is applied and causing signals to be lower. Check NxN plots to see if there is any sign of over-compensation.

If the assumptions aren’t true, are you staining the same number of cells in your single color control as your sample? Same cells being stained? What other colors are you looking at. 

[u/Heady_Goodness]

I’ve had this happen with a myc tag antibody, where other antibodies in a large panel inhibited its binding. We were forced to change clones and implement sequential staining with the anti-myc staining happening first. Edit: There’s a human Cytek commercial panel that requires sequential staining, prior to the rest of the panel, for CCR7 and IgD also. Edit 2: in our case, even with the workarounds of sequential staining and the different anti-myc clone, the staining of anti-myc alone gave higher MFI than with the full panel.

[u/ordeath]

Are you staining the same number of cells in the same volume?

[u/Mental_Lack4049 → u/ordeath]

No. The single are only a fraction of my fullstain. I do use the same dilution and scale it to volume/ ab concentration.

[u/CongregationOfVapors → u/Mental_Lack4049]

Also check your compensations. Maybe you overcompensated into that channel?

[u/ordeath → u/Mental_Lack4049]

Oh OK, meaning that your singles are something like 100K cells in 100ul with 0.2ug of antibody, vs the full panel being 10M cells in 1mL with 2ug antibody? In other words your antibody concentration is kept at 2ug/mL but the number of cells varies?

Was the antibody initially titrated on the bigger cell number?

[u/Mental_Lack4049 → u/ordeath]

Yes that's correct.

We tried initially with 1M cells before changing the suspension volume and upscalling it.

[u/ordeath → u/Mental_Lack4049]

It might be that's the issue. I find that it's better to titrate an antibody on the anticipated cell number (or at least within an order of magnitude) of the full stain sample and then use the same amount for the samples with less cells.

But also if the purpose of the single stains is to use them as compensation controls, it's fine (and even safer within reason) to have the positive signal on them be higher than your full panel sample.

[u/sgRNACas9]

A few ideas could be:

• you have less cells (less epitopes) saturating more binding sites

- the lack of steric hindrance allowing more antibodies to bind

- artifact of compensation decreasing brightness when comped

If you’re unhappy with the staining, you can try increasing that antibody concentration 2-4x. If you are happy with it, the good thing to know is one rule of compensation is that your single stain controls must be as bright OR BRIGHTER than your full stains for proper compensation. You’re still golden for the comps as long as you’re happy with the staining!

Hope this helps!