Fri Feb 02, 2018 12:22 am by tomash
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.