Is mass cytometry made for me? Is the analysis not too complex and only restricted to experienced bioinformaticians?
🔊Mass cytometry is a powerful tool for any immunologist. Our aim is to make this technique more accessible and understandable for everyone, by explaining it from our own user experience. Mass cytometry analysis is the ability to highlight immunological patterns and extract the most valuable information by focusing on a set of subsets, which are different in groups.
✏️ In a nutshell:
Mass cytometry is powerful discovery tool and is completing other techniques like single cell RNA-sequencing or flow / spectral cytometry. Briefly, sc-RNA-seq can help you design your antibody panel, whereas flow or spectral cytometry will validate the first result you found with mass cytometry.
The benefits of CyTOF in immunology:
CyTOF is tool for any researcher working in immunology, at least. With CyTOF, you will:
📈 easily increase the number of parameters you study 🚄 without spending too much time into panel design
👉🏽 remove autofluorescence and be more confident with the data you analysed
☀️ acquire more easily tumor samples (no autofluorescence)
💰 explore your research area in a more efficient way and use unsupervised mass cytometry analysis to discover new patterns .
Let CyTOF make you competitive in your field
Some research areas are still new to CyTOF. As an example, the number of CyTOF studies are very limited in diabetes, this technology has potentially a lot to bring.
#1 Design your panel properly
It might be hard to find the panel you would like and the markers you want to study. Before starting to dive into it, the two main questions are:
(1) which markers should I consider to study ?
(2) how do I design the antibody panel?
Get inspired by other work
(1) Choose relevants markers:
Since mass cytometry analysis is not new anymore, you can find some inspiration from other labs in your field. This varies a lot depending on your research area. As an example, in diabetes, mass cytometry has not been extensively used, so you have some space to create your own path. However, in cancer field, intensive efforts have been made the last years to reach a maximum of parameters and efficiently design it.
Some markers are being internalised very rapidly like CTLA-4, CD69 is also called the Very Early Antigen Receptor, so you might pay attention to its volubility. You can gather good ideas from the list of already published articles. We summarised their panels for you, so your job is easier.
(2) How to design the antibody panel?
You need to match the channels receiving less background with the dim expression markers to be sure you will receive true signals to interpret. There is also an online tool to help you design your panel.
Single-cell RNA sequencing: a more personalised idea of your samples
The most unbiased way to select the markers you would like to study would be to perform single-cell RNA sequencing on your samples. Later on, you also would be able to merge your findings with this technique, making your point stronger.
#2 Don't wait for the perfect panel: compensation is still possible
Mass cytometry has an incomparable strength over other techniques like flow or spectral cytometry: true signals. Except if patients or animals were specifically treated with heavy metals (this can happen in case of interventional radiotherapy in cancer for example), your samples won't need compensation. Acquiring tumor samples on a flow cytometer might be challenging, because myeloid cells express themselves some autofluorescence and might interfere with other signals. With mass cytometrty, the signals will be clearer and more easily interpretable.
As you know, some channels are giving only pure signals and some might contaminated with other signal from oxydation or from neighbours. However, don't spend too much time over-optimising your matrix. In any case, you can compensate it. this will remove unwanted signals and will guarantee better results. This is not a compulsory step, but you will be ensured that your results are not coming from an artefact.
#3 Realise that choosing your panel is being biased
You just found your panel suited for your analysis. You are in an exploratory phase, so you chose mass cytometry for the most unbiased approach. But choosing a panel is already being somehow biased. You can choose a panel more focused on lymphoid or myeloid, or a combination of both. At the end, keep in mind that CyTOF is not single-cell RNA-seq and you will be biased in some way.
#4 Minimise the batch effect
By freezing the antibody mix
If you have to analyse multiples samples, and you cannot stain them all in once, you can freeze the antibody mix. Just prepare some aliquotes of a stock mix solution. And thaw an aliquote from the batch once you are ready to stain your samples. In this way, you will minimise the batch effect linked to the staining.
By acquiring the samples from the same detector
As you may know, the detector in a mass cytometer cannot last forever. Each time you are using the mass cytometry, the detector is a bit damaged and often, more current is needed to analyse your cells. You can minimise this technical issue by booking the CyTOF some days in a row or grouping your samples on the same machine. There are also tools which were designed to reduce technical variability after acquisition if you think it is needed. However, those tools might also removed biological variability as well.
By applying a similar rate flow over your samples
The image you get by analysing your samples depends on the speed you are analysing it. Each cell should be well separated. A good balance between resolution and duration of te acquisition is a flow rate between 300-400 cells/s.
You can achieve such by following our calculation sheet while preparing your samples before acquisition.
👉🏽 Download our calculation sheet to easily see how much you should dilute your sample and the duration of the acquisition. This depends on the target flow rate you set.
#5 Check the quality of your samples
There are several tools to check the quality of your samples. You can also compare the intensity of expression of some markers you know should be constant between groups.
#6 Analyse your data in the most unbiased way and personalise your pipeline: use R!
The best advantage of mass cytometry is the number of parameters you can use to analyse your samples. With 40 parameters, you won't be able to represent every pairwise combination for marker. You need to analyse your samples in an unbiased way. One of the simplest way, but the least flexible and also the most expensive is to use common tools available online. But these services are expensive and not flexible. You can learn how to analyse mass or flow cytometry analysis simply by following our R training. You will need a couple of hours to go through the whole training and you will be able to use R. You won't spend time learning aspects of R you will never used. This training is going directly to the aim you are looking for.