2 August 2021

A new flow-cytometry-based protocol to measure single-cell protein expression in small samples

new protocol

An optimized protocol for simultaneous detection of fluorescent proteins and intracellular and surface antigens in the embryonic pancreas from the mouse. The protocol enables analysis of one pancreas per sample, thereby facilitating detection of biological variation and minimizing the number of experimental animals needed

In this very detailed laboratory protocol on how to label, count and sort cells from a very small sample (a single E11.5 embryonic pancreas), the authors, Nyeng et al. STAR protocols 2021, for the first time, designed a comprehensive procedure for analyzing the expression of both endogenous fluorescent protein, extracellular protein and intracellular protein in a very small sample.

The protocol published in STAR protocols, a journal with a very high standard for the level of detail. The manuscript went through a thorough review requiring substantial details and useful information for anyone wanting to setup a flow cytometry based experiment for the first time, (for instance, a table on which controls to include and a troubleshooting section).  

For some research questions such as ours, it can be essential to know the individual cell population in only one embryo

Pia Nyeng

The protocol was designed originally for a study published in 2019 (Nyeng et al, Dev Cell 2019), where the authors needed to analyze the protein expression level of P120CTN, E-cadherin, Pdx-GFP, and a pancreatic progenitor marker in pancreatic cells from a single mouse embryo. Essentially, they wanted to measure if there were both high and low P120CTN-expressing cells in one pancreas. Being able to analyze a single embryonic pancreas at a time without having to rely on tissue pooling reduced the number of experimental animals used, consequently saved both cost and time.

We had already published the method in the Nyeng et al, Dev Cell 2019 paper, and were subsequently approached by the lead editor from STAR protocols who asked if we would like to contribute a more thorough version for this new journal. Silja Heilmann (Semb group) and I have worked in parallel on another STAR protocols paper based on a computational method also included in Nyeng et al, Dev Cell 2019, which has just been accepted. Working with the team at STAR protocols has been more laborious than first anticipated, but we have learned a lot in the process, and I hope the result will be useful to many other researchers.

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The protocols that were available before this one relied on pooling from several embryos, where here we discussed thoroughly how to optimize the process, using advice from several other DanStem researchers.

Pia Nyeng