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Staining temperature

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sedejong

Participant

Posts: 7

Joined: Wed Mar 04, 2015 3:42 pm

Post Wed Mar 04, 2015 6:26 pm

Staining temperature

I am just starting with CyTOF, which I would like to use for analysis of my human PBMC samples. After CyTOF, I am thinking of further investigating interesting cell populations by flow cytometry, as that will allow me to measure my additional samples faster.

The standard CyTOF staining protocol here includes staining with antibodies for 45 minutes at room temperature, while for flow cytometry our lab always uses 30 minutes at 4 degrees. To be able to make a fair comparison between CyTOF and flow cytometry data, I would like to use a protocol that is as similar as possible.

However, I am afraid that increasing staining temperature for flow cytometry will 1) negatively affect my fluorescent dyes, especially the tandem ones, 2) increase background staining, and/or 3) increase cellular activity which could maybe change staining patterns or fluorescence etc. On the other hand, lowering the temperature for CyTOF would maybe lead to less efficient antibody binding or would require retitration of the panel.

Are these indeed points of concern, or are there other reasons to choose a certain protocol? And what do you think is best for me to do? Thanks!
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nlazarus

Contributor

Posts: 20

Joined: Wed Nov 13, 2013 7:28 pm

Post Thu Mar 05, 2015 5:33 pm

Re: Staining temperature

Personally, for both CyTOF and FACS, I stain at 4C. I haven't heard of staining at RT for CyTOF.

Nicole
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elinastar

Participant

Posts: 19

Joined: Sun Nov 17, 2013 1:46 pm

Post Thu Mar 05, 2015 8:35 pm

Re: Staining temperature

Hi,
We perform surface stains at room temp, for 40min-1.5hr. For intracellular staining we keep cells on ice for 1hr at least. Hope this helps!
Elina
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Fri Mar 06, 2015 7:00 am

Re: Staining temperature

Hi,

I would suggest u to perform few experiments: Incubate at 4 degree after Ab staining for 30, 45 and 1hr and Incubate Abs at room temperature for 30, 45 and 1hr (use FC block).
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Mar 06, 2015 4:32 pm

Re: Staining temperature

Hi,

The HIMC typically does surface staining and intracellular staining on ice (4C). I typically do 1hr for surface, and most intracellular staining for 45min or so. These time frames could probably be shortened a bit, but the timing is convenient (lunchtime, machine warm-up, etc).

As Komal said, you should probably just try a couple different protocols. Among other things, we typically do *not* use FC block or serum block, but I know there are several labs that do that on a regular basis.


Mike
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Jahangir

Master

Posts: 52

Joined: Sun Oct 29, 2017 6:34 pm

Post Thu Feb 01, 2018 5:50 pm

Re: Staining temperature

Hi guys,

Been reading this thread and I want to question the rationale for incubating the antibodies, both intra- and extra-cellular antibodies, at 4c?

What benefits are there in incubating at 4c rather than at RT? In my head, it just takes longer for the antibodies to bind to their epitopes but may reduce non-specific binding.

Many thanks,

Jahangir
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tomash

Contributor

Posts: 25

Joined: Sun Oct 19, 2014 10:15 pm

Post Fri Feb 02, 2018 12:22 am

Re: Staining temperature

Hi Jahangir,

Speaking from our perspective: for the surface stain (i.e. live cells), keeping the cells at 4*C prevents the cells from being metabolically 'active' during staining. Some antibodies that bind to proteins on cells will cause intracellular signalling cascades, and some proteins expressed on the surface will naturally turn over if left at room temperature. When we look for CD115 (the M-CSF receptor) on mouse bone marrow cells, the longer they are at room temperature, the more the marker is down-regulated. If we keep the cells on ice (for general handling and staining) then you prevent intracellular signalling and surface marker turnover. In the case of CD115, growth factors down-regulate when a cell leaves it's growth niche (in this case when we take it out of the bone marrow), but not if we keep it cold.

Surface staining cold probably does mean that antibody binding will take longer, but we only stain for 30 min at 4*C for surface staining -- not a huge inconvenience.

Intracellular staining is a little more difficult answer. Once a cell has been fixed (normal fix, or fix/perm), they don't need to be kept cold to prevent metabolic activity, so room temp is fine. However, the kind of reagent being used for fix or fix/perm is the critical factor. Many probably work better at RT than 4*C, but unless you know what the reagent is, and have tested the reagent in both conditions, then it's best to just do what the company's protocol says. You can get some pretty huge differences in staining with some reagents - I recall Pratip showing some examples at CYTO one year with great vs absent labelling due to changing the staining temperature. The best example is probably the FoxP3 fix/perm buffers. Because FoxP3 is a transcription factor, the kits use some magical ingredient to free up the FoxP3 binding site from DNA. If it is an enzyme/compound that is temperature sensitive, then that will be the most significant factor in deciding the temperature. Just a side note, most of the FoxP3 or transcription factor kits have the magic ingredients in the fix/perm buffer, but not the perm wash buffer (which is usually just saponin) -- but it's safer not to make assumptions unless you actually know.
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JamesW

Contributor

Posts: 37

Joined: Tue Nov 21, 2017 6:59 am

Post Fri Feb 02, 2018 1:10 am

Re: Staining temperature

Hi Jahangir,

Its also important to note that some targets cycle on and off the surface so by surface staining at higher temperatures you can stain a larger fraction of the total pool, many chemokine receptors function like this.

Mario Roederer's lab published a paper on this and basically suggest using 37C surface staining for chemokine receptors
J Immunol Methods. 2003 Aug;279(1-2):199-207. Optimized lymphocyte isolation methods for analysis of chemokine receptor expression.
https://doi.org/10.1016/S0022-1759(03)00186-8

For Cytof specifically, Ewan Newell uses a similar method of a 37C pre-stain for certain chemokine receptors followed by the rest of the staining at 4C.
Immunity. 2016 Aug 16;45(2):442-56. doi: 10.1016/j.immuni.2016.07.007. Epub 2016 Aug 9. A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures.
https://doi.org/10.1084/jem.20160897

I'm in the process of testing out my own panel and can say that most the chemokine receptors benefit from this, as does CD45RA. However, as pointed out earlier some markers may actually get degraded so you have to test it on a case by case basis. I'm edging towards a 30 minute 37C pre-stain followed by staining at 4C for 1 hour.

Best,
James
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Tue Feb 06, 2018 4:48 pm

Re: Staining temperature

Hi Everyone,

Great discussion! Just to add to it, we typically fix cells before staining, so we usually stain at 37C under the assumption that this will be faster and easier. The few times we've done live staining, we have performed surface staining it at 4C to prevent signaling responses and receptor internalization, followed by intracellular staining at 37C.

I do think that the most important thing is that you titrate antibodies under the conditions you plan to use. With CyTOF this is pretty easy, however, I would start by just running your current panel at 4C and then, just re-titrate the things that don't look good. If you already have a protocol that works well by fluorescent flow, I would stick to that (even if it means retitrating), since the antibody concentration will be much easier to figure out than the effects of temperature on the biology of the specific antigen-antibody interactions that you're studying.

Best,

Greg
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Jahangir

Master

Posts: 52

Joined: Sun Oct 29, 2017 6:34 pm

Post Tue Feb 06, 2018 5:47 pm

Re: Staining temperature

Dear Tomash, James and Greg,

Many thanks for all your replies, they were most definitely helpful. I would just like to ask, the temperature for staining both intra- and extra-
cellular markers shouldn't matter as much if the cells are fixed?

Many thanks,

Jahangir

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