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A 14 colour antibody panel: developing a tool and demonstrating a process

One of the frustrations I have with Flow Cytometry is when companies present their amazing new findings at conferences, and it’s quite often about TMB cells. In my case, I work on these cells perhaps 20% of the time. The rest of the time I work on cells from other parts of the human body – bone marrow, lung, bronchoalveolar lavage, spleen – and in diverse animals such as mice, rats, and even sparrow, chicken, and mosquito.

The lymphocyte work is only a very small part of what I do, so I was eager to develop new Flow Cytometry techniques outside of that range. This brought about the multicolour antibody panel for mouse bone marrow stromal cells which I will be discussing at the Flow Cytometry Congress. The process provided important insights into the process of looking for and analysing rare cells.

Developing a 14 colour panel for mouse bone marrow stromal cells

I was at a conference with a colleague, viewing the Flow Cytometry work of people looking to get the best combination of fluorophores and antibodies onto certain cells. It was predominantly TMB cells panels: human or mouse immune cells. We were discussing the fact that we investigate more than simply immune cells and have people working on stem cells and RIN-m cells, and that we wanted to provide people with a tool to look at very rare cells. What if we developed a panel that combined every marker that had ever been proven to exist on a mouse stromal cell?

We began searching through the literature and seeing that there was a lot of differing information about the area, and even debate centering around whether these cells existed. Nevertheless, we found one study that said that certain biomarkers were expressed in cultured stromal cells.

But we have found in fresh cells that some of the markers are differentially expressed, or not expressed at all. We slowly started working up the protocol, adding fluorophores and changing the way we processed the cells and the protocol we used as we found we just weren’t seeing the cells we expected to appear.

My experience of developing this 14-colour panel is that it can be difficult to get the best out of experiments. One needs to think about overall experimental design: sample preparation and background subject knowledge, and the data analysis on the other side. Taking all these factors into consideration builds a more holistic picture than can improve data validity.

The main challenge we have faced is the uniqueness of these cells: they are extremely rare (we must run a lot of samples to see a population at all) and they are also not well characterised. We have had difficulty deciding on what markers and antibodies are the best to use, as the literature varies significantly. One part of the literature will highlight one as being most important, whilst another will discard it completely.  Essentially, we have been trying to answer the question: what is the truth about these cells?

More than a panel

We wanted to provide a tool for people to use effectively in their Flow Cytometry work with rare cells. Building a multi-colour panel takes months; developing something with 14 colours can take up to nine months to complete and validate. What we hoped to produce was not just a panel but also the techniques to validate it, so people don’t have to spend time validating all the antibodies when looking for their cells. They can simply copy what we have done and edit their own process, even if they only use part of the panel for their work.

What we hope to do when published is to demonstrate that it is really a whole process and not just a panel. We also want to show the validation techniques and announce the features that we used to get the best out of the data.

 

Jane Srivastava is the Flow Cytometry Facility Manager at the South Kensington Campus of Imperial College London.

 

Expert speakers will discuss the latest technological developments and applications of flow cytometry on cellular analysis and in cancer diagnosis and treatment at the Flow Cytometry Congress. Take a look at the agenda

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