Designing a panel to analyse mouse haematopoietic stem cells

Flow cytometry core facility managers play a very important role in helping scientists with their research and maintaining valuable flow equipment. We are pleased to feature Dr. Steffen Schmitt, the Head of the Core Facility Flow Cytometry at the German Cancer Research Centre (DKFZ) in Heidelberg.

Dr. Schmitt spoke to us about the challenges in flow cytometry, the tasks involved in running a core facility and the importance of cell sorting. He also described his recent work on optimised multicolour immunophenotyping panel (OMIP-059)¹, in which he and a team of researchers designed a 16-parameter panel to analyse mouse haematopoetic stem cells and their immediate downstream progenitors.

1. As head of a flow cytometry facility, what expectations do researchers have of you?

There are many different expectations a core facility has to meet nowadays. It also depends on who you are asking. We have users, who are mainly postdocs, technicians and Ph.D. students. We also have group leaders, department heads and management.

Besides, we are in contact with collaborators, other core facilities and are keeping contact with companies in the field. Each of these gives us different tasks. If you ask our users, for example, they want us to offer high-end services, to have enough capacity and to organise the daily work life and quality control.

They ask for cutting-edge and state-of-the-art equipment, and their maintenance. In the end, it should be as (cost-) effective as possible. We are also asked to provide training and education for students. Our internal training is attached to our in-house Ph.D. program and together with our colleagues from European Molecular Biology Laboratory (EMBL), we organised an European Molecular Biology Organisation (EMBO) course for high-end cell sorting.

Also, we are a part of the iBiology online video platform. Group heads and postdocs also ask for scientific support. We are often involved in the planning of experiments, which these days mostly involves help with panel design for multiparameter analysis. In the end, we also help with data analysis using the new tools which have come up in the recent past.

We often represent our institution through our technology in various places like conferences, public events like the Science Night in Heidelberg, open door days, and in special events like the so-called Unistem day, where life science students come to learn things. We also partner with companies to organise workshops and seminars.

2. What are the challenges in flow cytometry?

It may be surprising, but I’ll put flow cytometry education at a very high rank for the following reasons. In theory, it is a very simple technology—you just put a stream of single cells in front of a laser and record the data.

In daily life, however, what I’m observing is that many people underestimate the combinatorial possibilities that are available now with the different lasers and fluorochromes, which make flow cytometry relatively complicated.

As a core facility, we have the responsibility to ensure quality in terms of instrument performance, experiment layout, and subsequently, of the data which are generated. When you look at publications, for example, you often don’t find enough information to be able to reproduce the results of experiments, which could be for several reasons.

Sometimes users just might not be aware of the importance of certain information, whereas in other scenarios it might be a lack of information or education. This is something we need to work on.

3. How can a company like BD help the flow cytometry community meet these challenges?

BD Biosciences and other companies are addressing this problem by organising lecture series and courses. You also have these high-end user meetings (e.g., in Heidelberg), where you invite specialists in the field of flow cytometry and discuss topics with them.

Besides flow cytometry, you also offer tools for sample preparation, and I feel that is mandatory. Flow cytometry has moved out of the realm of immunology where it is relatively easy to get single cells out of a lymphatic organ.

Nowadays people are studying tumours, epithelial cells, brain, guts and cells from other tissues which are much more demanding, so it is very important to start with a good-quality sample. These days we have a spread of topics in flow cytometry like conventional, imaging, spectral and mass cytometry for analysis and cell sorting.

While cell sorting with the conventional flow is already state of the art, it would be interesting to see a combination of all topics with cell sorting in the future. The most important thing with cell sorting is that it allows us to have access to single cells, which lets us analyse them in ways we couldn’t before.

With all the new technologies to study single cells that are coming up, the importance of cell sorting is continuously increasing.

Yet another challenge is about integrated workflows for protein as well as genomic investigations, and I know that BD is heading in that direction. At the moment, I have the feeling that at least in our institute, Ph.D. students have to be the managers of their single cells.

They collect samples at the animal facility, prepare them for flow cytometry, do the genomics, get the sequences out and then do all the bioinformatics. Each of these requires the help of a different specialist. So, having an integrated workflow would be very useful.

Another word that I haven’t used is microfluidics. This might impact the field of single-cell analysis in the future. This is also related to another topic I have in mind and that is waste disposal.  This is important if you consider the amount of liquid waste we are producing and the need to manage that according to the regulations.

With microfluidics, you produce much less waste and I think this has been underestimated so far. It is okay if you have only one or two instruments but for bigger labs, it could be more demanding. It would be very useful if companies like BD could address this topic.

4. Can you talk about your research on haematopoetic stem cells?

The haematopoetic system is a very fascinating system to study as it can re-populate an entire system starting from just one cell. Even after working on so many kinds of cells, this is still unbelievable and fascinating to me.

The idea to establish such an OMIP is nearly as old as the publication format, but it took us a while to find the capacity and the time to do it. In the end, we were quite surprised that no one else published it before, as several groups are studying the haematopoetic system.

I work in a cancer centre and we are interested in more than just the haematopoetic system by itself. A few years ago, there was this theory of cancer stem cells by Irwing Weissman and others.

It turns out that there are fascinating similarities between the haematopoetic and cancer stem cells (e.g., if you look at the dormant haematopoetic stem cells, they are often sitting in a niche and being somehow protected from the environment).

One idea is that cancer stem cells could as well be sitting in a niche that is protecting them from drug treatment like chemotherapy. Both cell types are also very rare. For a long time, it was thought that cells in a tumour are relatively homogeneous, but now, we have learned that it is the exact opposite.

When studying tumours at the single-cell level we have to consider the fact that they are composed of several different subsets of cells with altered properties. We need to study deeper and understand these subsets. There are several new findings about haematopoetic cells now.

If we can understand better how they behave, we can transfer that knowledge also to cancer stem cells. This will help us to assess new treatment approaches, learn how to overcome drug resistance and so on.

The main challenge is mostly to get enough cells, sort them and go back to answering the questions I have already talked about.

5. How did you design the OMIP used to analyse these haematopoetic stem cells?

We tried to achieve different things with the 16-parameter (14 colours plus 2 scatter) OMIP. The first thing was, of course, that we wanted the panel to work with both depleted and non-depleted bone marrow, as there are different ways of preparing the sample.

For some experiments, one starts with depleted lineage positive cells while for other layouts, one can start from the whole bone marrow. Not all our users need to go down to every single progenitor level. For many experiments, it is sufficient to have Lin⁻Sca-1⁺c-Kit⁺(LSK) cells, which already contain an enriched fraction of stem cells.

So, we thought we should try to build a panel such that not everybody has to use different antibodies and different panels. Those that use only 5 colours can use the same combination, as can those who want to study the system deeper using 10, 12 or more colours.

Another thing is that many studies deal with transplantation experiments—putting cells from one mouse into another. There, we often use a polymorphism of different antigens like the common lymphocyte antigen CD45 which comes in different variants (CD45.1 vs CD45.2) which are allowing us to discriminate later on between donor and recipient.

To make it more flexible and interesting, we had the idea of incorporating a fluorescent protein-like green fluorescent protein (GFP) as transgenic mice expressing GFP are used in haematopoetic stem cell research quite often. Based on these prerequisites, we used our experience and knowledge of which fluorochrome combination promises a good sensitivity and which we should avoid, for building this panel. We are happy that we succeeded and got it published.

So, it is now a standard, at least for our groups at the DKFZ. The panel can directly be included in their research experiments, backed up with this publication as the citation root.

More on this topic: Flow cytometry panel design

6. How did the fluorochromes perform?

The two dyes brilliant ultraviolet (BUV) 737 and brilliant violet (BV) 480 are classified as roughly having the same brightness and we used CD16/56 BUV737 and CD45.2 BV480, so we first thought it may not make a big difference. But then, we saw in our experiments that there is a need for optimisation to improve the separation for these two parameters.

We switched BUV737 and BV480 to other antigens and finally, we ended at BUV737 CD45.2 and BV480 CD16/56. CD45.2 is in combination with CD45.1 to discriminate between donor and recipient. Both markers are expressed on different cells and we only needed a “yes” or “no” information.

Therefore the separation in this combination was less important. For CD16/32 it was different (e.g., on granulocyte-macrophage progenitors (GMPs) CD16/32 is coexpressed with CD34). And to find a fluorochrome combination with good separation, we took a deeper look into the dye spreading matrix of our instrument to redesign the panel, and ended up with BV480.

This showed us that flow cytometry can still be very demanding in certain steps and is not as easy to transfer theory into practice, even for people with a relatively high level of knowledge. For example, when we put the published OMIP into the BD Horizon ™ Guided Panel Solution, the output revealed that four antibody-fluorochrome combinations would not be matching.

Again, one of them was BV480.  Other differences were with the LSKs – the Sca1 and the c-Kit- where we used very bright dyes. These are very strongly expressed antigens and the theory would suggest that we use less bright dyes for them.

However, they are on the top of the gating hierarchy and we are also referring to intermediate populations in between. We also wanted to get the best separation for them to enable us to go further. Nevertheless, the published combination worked in our hands, which clearly shows that it is very important to test every panel for its performance before taking the final decision.

By designing a panel the researcher should always ensure that there are good arguments for why one should use a certain antibody-fluorochrome combination and not another, irrespective of how it was built up. In our case, BD Horizon ™ Guided Panel Solution was mainly supporting our decisions.

7. What do you think of ultraviolet dyes?

I grew up with flow cytometry during the 1990s.  Back then, ultraviolet (UV) was used only for special applications like calcium influx and side populations, which used to be the gold standard to analyse stem cells. The point I’m trying to make is that in those times, it was quite easy to answer the question of whether one needed to invest in a UV laser or not.

Nowadays nobody does side populations to study stem cells regularly and for calcium influx, there are other fluorochromes available that do not need the UV laser. UV lasers are relatively expensive and for a while, besides the already mentioned reasons, there was no real need to have a UV laser built in the instruments.

Those who had one were struggling a bit because for the money invested, they could only get one or two more parameters, mostly for use in DNA staining.

BUV dyes, of course, take the UV laser to another level. Today, with these fluorochromes, you can use the UV laser as just another laser line, possibly even more as it has a very large spectrum ranging from 380-390 to almost 800 nm. You can put in a lot of additional fluorochromes.

The UV laser capacity for fluorochrome discrimination allows now for a 20–30 parameter analysis.  So, even if it is not a cost-neutral add-on, it gives you possibilities that you will not find anywhere else. Only BD offers both the UV laser and the broad spectrum of antibody-fluorochrome conjugates for it and I don’t want to miss it anymore.

More on this topic: Making the most out of the ultraviolet laser

8. How important is it to perform single-cell sorting and analysis under the same conditions?

I’ve already mentioned the importance of single-cell sorting. I think it is one of the most significant advances made by science in the past decades. It is a prerequisite for so many applications that use single cells.

If you look at the publications, there has been an exponential increase in the number of studies on single cells in the past 5–10 years. Without cell sorting, all this would not have been possible. Now different technologies are coming up that also use single cells.

Cell sorting and analysis under the same experimental conditions is a nice thing to have. It can speed up things and you can perform tests like what we did in the OMIP. You can analyse on an analyser and quickly transfer that to a cell sorter.

But I think it is not a must-have. Even if you had two different systems, you could still succeed but maybe you would need more time (e.g., for panel redesign, additional testing and assay development). It also adds more flexibility to your experiment and reduces continuity.

When it comes to adding one more parameter to your panel, then it could be a big advantage to be on a comparable system. What we want is at the end to study and understand the heterogeneity of biological systems at a single-cell level.

Browse our selection of BD Horizon™ BUV dyes.

The views and opinions expressed in this interview are solely those of the interviewee(s) and do not necessarily reflect the view, official policy or position of Becton, Dickinson and Company. 

The opinions and clinical experiences presented herein are for informational and educational purposes only.

The interview is 2 years old and as such doesn't contain information on more recent products and insights.