Table 68.

Selection of important pitfalls and top tricks for multimer generation und usage

Theme	Solution	Pitfall	Top Trick
TCR down regulation	Staining at 4°C reduces TCR internalization (only MHC I multimers)		+++
TCR down regulation	Staining at 37°C increases TCR internalization; Accumulation of signal for MHC II staining		+++
TCR down regulation; Dim signal	Use bright fluorophores for best signal-noise ratio (PE; BV421; APC)		+
False positive TCR stain	Multimer double stain with two different conjugated Fluorochromes		++
False positive TCR stain	Staining of unspecific cells (especially B cells via PE conjugated MHC multimer)	++
False positive TCR stain	Dump channel and live/dead discrimination; Pregating for CD3 and/or CD8/4		++
False positive TCR stain	Certain CD8 mAb clone interfere with MHC binding, leading to unspecific signals	++
Reliable TCR binding	Multimer binding is not as specific as mAbs; Controls for peptide unrelated MHC-TCR binding	+
Multimerization level	≥ 3 binding partners are sufficient; questionable improvement by using Pentamers and higher multimerization levels due to unspecific binding	+	++
Reversible Multimer staining	Unaffected T cell function after dissociation of multimers		++
Reversible Multimer staining	TCR-pMHC off-rate measurement		++
Rare cell detection (naïve T cell compartment)	Pre-enrichment methods (MACS, previous enrichment sort)		++
Co-receptor blockade CD8/4	First stain with the multimer for 25min, then apply Ab stain for 20min (second multimer for double discrimination can be included here)	++	+