I am working on characterizing cellular subtypes in PBMC. I never seem to quite get a good positive population for CXCR5, despite conducting titrations for the same. Is there a special way of staining for these receptors? I.e staining for CXCR5 first before adding other antibodies...
Most people would stain at 4 degrees which is what i can imagine you do,. However there is a simmering debate that actually staining at RT could be better then switch to rest of the staining at cold condition (4 deg)
I have attached a string of discussion from Purdue University Cytometrty laboratories between Mario Roederer replying to Stuart
Hi Stuart:
many chemokine receptors recycle continuously through early endosomes -- so if you freeze the endocytic pathway you can only address the fraction that is on the surface. If you stain at RT (or even 37C) you can hit a much larger fraction, and get better signal. This also true for some molecules such as the FAS-ligand.
We looked at this fairly extensively, and published optimal protocols for a large range of CCR's: See Berhanu, D., Mortari, F., De Rosa, S. C. and Roederer, M. (2003). Optimized lymphocyte isolation methods for analysis of chemokine receptor expression. J Immunol Methods, 279, 199-207.
In general, we do all of our staining at room temp without any drugs to block capping -- we have never seen a problem. For many CCR stainings (like CCR7, eg) we stain at 37C for 10-15 minutes, then shift to RT and add the remaining antibodies for another 15 min. We rarely stain on ice anymore.
Even for antibodies that might be carried inside the cell -- the fluorescence remains with the cell, so this isn't a big problem. Exposure to proteolysis is slow, and doesn't happen for many surface receptors that are internalized anyway.
Gerhard is correct in noting that staining at higher temperatures accelerates the kinetics, allowing you to reach saturation with lower concentrations of antibody. It is important (and economical) to titrate your antibodies under the conditions you will be staining with experimentally. In general, staining at room temp allows you to use about 1/4th the concentration as you would use on ice; and at 37C, 1/2 that at room temperature. But that's a generalization -- do the titration!
We have never seen viability problems with lymphocytes stained on ice. One issue we saw years ago was that if you chill lymphocytes, they do not respond as quickly to stimuli (so, for example, calcium flux experiments don't work well). But if you stain at room temperature, their signaling is fine. Longer-term functional assays (6+ hours) seem to be unaffected by chilling to ice temperatures.
regards,
mr
On Feb 14, 2011, at 1:15 AM, Stuart Berzins wrote:
> Some staining protocols recommend staining at room temperature instead of at
> 4 degrees c. One lab here recently stained for chemokine receptors and found
> they only got positive staining if it was performed at room temp - stianing
> on ice didn't work. That is fine, but they also notivced tha t some other
> surface stains they used were brighter than normal. They are now
> comntemplating doing all staining, surface and intracelluar at room
> temperature. My advice to them was to keep to ice because it slows the
> cyclining of surface antigens beteen cell membrane and intracellular
> compartments. As such, 'high' staining may not be accurate staining of cell
> surface expression. They are skeptical about this. Is staining at room temp
> fine, or is it better to keep it at four degrees?
Hi Ian
Could you give us details of your staining protocol to help you?
Chemokine receptors have seven transmembrane domains, all of which are fluid and moving around in the cell membrane. As a result, the three extracellular loops, to which antibodies must bind, are also moving and this motion can change the orientation of antibody binding sites. In addition to movement within the lipid membrane, chemokine receptors can undergo internalization and endocytic recycling, which can be induced by changes to the cellular environment during cell processing. All these factors can obscure the antibody binding epitope on the receptor and result in no staining.
Staining live cells at physiological temperature (37 °C) is one technique to avoid some of these potential pitfalls. This allows for chemokine receptors to remain fluid, increasing the likelihood that antibodies will detect the correct epitope conformations. It also allows internalized receptors to be recycled back up to the cell surface. Staining at 37 °C can improve the detection of many chemokine receptors by flow cytometry on live cells.
Reference
https://www.rndsystems.com/blog/heat-detecting-chemokine-receptors-live-cells
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