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Can I use bead enriched samples?
Answer Yes, you can use bead enriched samples for CyTOF analysis. Most of the beads used for enrichment are not going to affect CyTOF analysis however there are a couple of things to consider. Some beads may be quite sticky and cause clumping of cells which in turn will decrease efficiency of sample preparation and may also cause clogging of the mass cytometer during acquisition. If the sample contains a lot of the beads they may accumulate on internal parts of the machine and therefore affect the analysis. If it is possible it may be best to do negative selection with the beads or try FACS sorting the cells.
How long will the acquisition take?
Answer There are a few factors involved when it comes to the acquisition time. In general, acquisition is much slower in comparison to flow cytometer and varies from 100 to 750 events per second. This in turn gives 360,000 to 2,700,000 events per hour. The acquisition rate will depend on the quality of the sample. The more metal contamination, debris and overstaining present in the sample the more we will need to decrease the acquisition rate. Also, we will have to acquire sample slower if the cells are sticky or big due to clogging issues. Once we know how much we can acquire it will be important to think how many events per sample we will need to analyse, bearing in mind that the count for the smallest population of interest should not be lower than 300.
How many samples can I barcode in one batch?
Answer Barcoding Kit supplied by Fluidigm allows for combination of up to 20 samples. It relies on the use of 6 different Palladium isotopes to create 20 various barcodes consisting of 3 isotope code.
Do I need to use specific slides?
Answer No – any microscope slide will work as long as it is made of glass.
How thick should I cut my sections?
Answer Standard 4-7 μm thickness is ideal. Thicker sections are likely to contain overlapping cells which may cause problems for accurate segmentation.
Can I use FFPE tissue?
Answer Yes, both FFPE tissue and frozen tissue can be used successfully with the Hyperion.
Can I use CyTOF antibodies for Hyperion staining?
Answer Antibodies that work for CyTOF staining will not necessarily work for Hyperion, particularly if you are using FFPE sections. However, in some cases, the antibody clone may work in both – we recommend that you check the antibody clone information to see if it is likely to work for normal immunohistochemistry before attempting to validate for use in the Hyperion. If no information is available to suggest whether a suspension CyTOF clone will work but there are alternative clones available known to work in immunohistochemistry, then we would recommend using these alternative clones first as there is a greater likelihood of success.
How do I know what concentration to use?
Answer Antibody titration is a key to optimising a good panel and it is important that you check different dilutions in your tissue of interest to make sure that staining is optimal. If you know an antibody works for immunohistochemistry in your tissue this can be used as a rough indicator of where to start your titration. However, it is likely that the antibody concentration will need to be higher for use the Hyperion compared to standard immunohistochemistry. This is because, in general, secondary antibodies are not used for Hyperion staining so the signal will not be amplified.
What controls will I need?
Answer In cases where you are unsure if or where your markers will be expressed, positive and negative controls are essential. The best positive and negative controls will be dependent on your markers of interest. In most cases, ‘minus one’ controls are not necessary but they can be used in cases where you are unsure whether a signal is due to spill-over in a particular channel.
How long can I store my slides for?
Answer Slides stained with metal-labelled antibodies will remain stable for years at room temperature if stored correctly. Slides should be stored in a dry, dust-free environment. Cardboard boxes should be avoided. The best choice of container would be a plastic slide box.
How long will my samples take to run?
Answer Sample running time is mostly dependent on how many regions of interest (ROIs) are required for each section and how large each ROI is. It will take approximately 3 hours to ablate a 1mm2 ROI. Set up for ablation also takes some time. This can vary depending on factors such as whether co-localisation of images is required. Set-up time is also dependent on how long panoramas take to generate. If possible, we recommend cutting 2-3 sections per slide if space allows. This reduces slide set up time as it takes time to change between slides.
Will I need to compensate my data?
Answer Spill-over with imaging mass cytometry is minimal and is affected even less than suspension CyTOF. However, spill-over can still happen. In most cases this can be minimised with careful panel design and titration of antibodies. However, it is still possible to compensate imaging mass cytometry data using similar methods to those used for suspension CyTOF.
How long does the staining take?
Answer Typically, staining is done over two days with antibody staining at 4°C overnight. The first day of staining takes approximately 4.5 hours and the second day takes approximately 1.5 hours. These timings are based on protocols recommended by Fluidigm and may vary if alterations to the protocol are made.
Does Hyperion have a microscope so I can see my sample?
Answer The Hyperion is equipped with a camera which will produce a 20x magnification image of your slide. This camera can be used to create an image of a particular area of your slide – this is your ‘panorama’ from which you can choose specific areas to ablate. However, no other magnifications are available and the live slide image only covers a small area of the slide at a time. If a larger, more detailed view of your sample is needed to choose regions for ablation the we recommend co-localisation of your slide with a slide-scanned image from a serial section, or an image of your section taken on another microscope.
Do the files need processing?
Answer Hyperion data is exported as an MCD file which can be opened directly in certain programmes, such as MCD viewer without processing. For analysis in other programmes, such as CellProfiler, files should be in TIFF format. MCD viewer can be used to process your images into TIFF format, as well as other formats, so that they can be imported into other programmes for downstream analysis.
What software do I use for data analysis?
Answer Different software packages are available for the analysis of imaging mass cytometry data. Most commonly, Ilastik and CellProfiler are used for pixel classification and generation of cell segmentation masks. After generation of a cell segmentation mask to convert the image pixels into single cell events, data can then be analysed using HistoCAT or HistoCAT++, which were specifically designed for analysis of imaging mass cytometry data. For more information, see the ‘Hyperion data analysis’ section.