Tips & Tricks: Temperature and CCR7 clones

As discussed last week, human CCR7 staining is highly dependent on temperature during the incubation. Today we'll look at the behaviour of three different clones--G043H7, 3D12 and 2-L1-A--at different temperatures. The first clone, G043H7, is available from BioLegend, while the 3D12 is sold by ThermoFisher and BD, and 2-L1-A is marketed by BD as their superior clone. I happen to have G043H7 in AlexaFluor 700 and the other two in R718, dyes that are spectrally similar, so I'll use those for this comparison. That's not fair to the G043H7 because R718 is much brighter than Alexa Fluor 700. I think, though, it's useful to see how the difference in fluorophore also plays into the selection of antibody for staining.

For today, I'm using 10^6 cryopreserved human PBMCs. They're stained with CD3 StarBright Violet 475, CD4 StarBright Violet 570 and fixable viability dye eFluor780. I've blocked with 3% mouse serum plus 3% rat serum. Staining is for one hour in PBS with 2.5% FCS and 2mM EDTA, using the manufacturer-recommended test of 5ul, which is a bit much for some of these at the higher temperatures. Also, the G043H7 is at 400ug/ml while the other two are supplied at 200ug/ml. After staining, the cells were washed and fixed with PFA.

The temperatures being tested are:

• 0C (on wet ice)

• 4C (in the fridge)

• 21C (in the dark at room temperature)

• 37C (in the incubator)

The master mixes are pre-incubated at the target temperature for 15min prior to staining.

A note on the unmixing: I'm treating the Alexa Fluor 700 as if it were R718 for the purposes of unmixing, which is not correct. I'm doing this because I'm being lazy and want to have all the data in one experiment with the CCR7 in the same "channel" as if it were conventional flow. If you look at the normalized spectra in the plot below, R718 is blue and AF700 is purple. They both peak in R4, with similar profiles across the spectrum, but there's a very slight difference in the far end of the violet that will manifest later as an unmixing error.

Spectra of the panel:

Gating strategy:

As mentioned in last week's post, incubating at 37C can cause cell death, particularly in fragile cells like cryopreserved PBMCs. That shows up clearly when we look at viability staining on all cells (ungated).

At 37C, the monocytes are starting to die (and perhaps engulf debris), as we can see in the scatter profiles:

All right, the part we've been waiting for:

What can we see here? The G043H7 and 2-L1-A clones get better up to 21C, but staining with the 3D12 clone doesn't change as much. Remember that R718 is a lot brighter than Alexa Fluor 700, so the G043H7 clone is actually quite good. There can be a bit more background with some Alexa Fluor 700 conjugates, and that's the case here. At 37C, there start to be artefacts, probably due to non-specific binding and dying cells. Also, you can see the consequence of treating AF700 as if it were R718 here (the G043H7 is skewed downwards versus CD4 StarBright Violet 570).

Let's put some numbers on that.

Gated on CD3+CD4+ cells:

You're going to get different interpretations of the biology depending on which clone and temperature you use.

It's not just the CCR7 staining that behaves this way, although CCR7 is more dramatic.

Suggestions: generally, if you want the best staining, go for room temperature. If you need to keep the cells cold, say for sorting, use the G043H7 clone. Use a bright fluorophore. If you're using fresh blood, you can probably stain at 37C. Titrate your antibodies under the staining conditions you intend to use.

I'll point out that OMIP-69 and OMIP-102 both used room temperature staining. It works well.

Reagents used:

CCR7 G043H7 AF700

CCR7 3D12 R718

CCR7 2-L1-A R718

CD3 StarBright Violet 475

CD4 StarBright Violet 570

Fixable viability dye eFluor780

BioLegend Fixation Buffer