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This month the MRC WIMM hosted the annual Regional Breast Cancer Academic Day, an event that brings together breast cancer teams from across the Thames Valley region. This year’s symposium involved the participation of over 70 attendees, including nurses, clinical trial managers, researchers and doctors.
Answer Slurm job profiling and top tips.
Answer Make sure you comply with the prevention of sample contamination included in the section for contamination. If you follow all the rules for minimising contamination your samples during sample preparation think about what happens with your sample before. If these are patient samples there may be a few things to consider. Please have a look at the list below organized according to metal: Iodine – iodine solution is used as disinfectant during surgeries, some dyes used by pathologists may contain it as well Barium – may be present on reusable parts of medical equipment, rarely patients are given contrast for some medical assessments which contain a lot of this metal Platinum - Cisplatin is one of the most common cancer treatments but it is also used for Live/Dead discrimination in CyTOF experiments. If patients that you collect samples from were treated with Cisplatin you will need to use alternative Rhodium staining for CyTOF Other – example being Yb, Sn, Pb can come from environmental contamination, water, solutions that may have contact with the sample before it gets to you
Answer When picking antibodies for your panel rules are very similar as for flow cytometry so you will need markers to discriminate your populations to find the population of interest and markers of interest that will give you answers to your research question/help you prove your hypothesis. In order to do this, you can use all information that is available in literature or from your previous research, including not only FACS and mass cytometry but also other techniques like immunohistochemistry, mass spectrometry. Antibodies that are used for flow cytometry and work well for you should be your first choice. However, the antibodies that you are interested in but you haven’t try in flow cytometry or even are not available as mass/flow cytometry antibodies may still work for your CyTOF experiment. Always bear in mind that it is very important to validate your antibodies so you and later your reviewer will be convinced that they are specific and the staining is free of artefacts.
Answer There are two options available for you. First, is to buy purified antibody in buffer containing additives and use kit to purify it (Abcam GOLD BSA Removal Kit – removes most of additives, not only BSA). Second, buy antibody conjugated with fluorescent dye that has an anti-dye antibody available and use two step staining.
Answer In order to validate your antibodies, you will need a good positive and negative control for each antibody. It would be ideal if you can have a heterogeneous sample containing populations which you can easily discriminate and include some that express and some that do not express the particular marker.
Answer Antibody validation is a crucial aspect of panel development and therefore it is very important to have appropriate controls. In some cases, this may be challenging if the marker is highly expressed in most of the cells (e.g. metabolic enzymes) or on the other hand its expression is very low. An option you may need to consider is looking for a knockdown or overexpressing cell line respectively.
Answer A standard mass cytometry protocol assumes starting with 1 to 3 million cells. Although if the population of interest is small you may need to start with more cells or think about previous enrichment of your samples. On the other hand, there are samples where recovery of a million of cells is an unrealistic dream then you may want to think about creating a bulk out of other cells that will ensure that the cell recovery after an extended staining protocol is still good.
Answer If the cell count for the sample to be analysed on CyTOF is very low it will be beneficial to use different cells to create a bulk for staining. When choosing cells that will be used as a bulk it is important to remember to pick cells that will be easily available every time you need them, easily distinguishable from your sample and will not catch/‘eat’ antibodies you use for staining. A good option also would be to barcode and pool your samples together. This is probably viable for samples with cell counts well below 1 million down to 100,000.
Answer To detect really rare populations, you need either to acquire a lot of events, or enrich the samples for the population of interest. Acquiring high numbers of events in order to have reasonable count for the rare population of interest is highly inefficient and may be costly on CyTOF, thus the best option would be to enrich the samples for the population of interest by FACS sorting or using bead-based enrichment methods.
Answer Yes, you can use FACS sorted samples for CyTOF analysis. The important issue you may want to consider if you are going to use the same markers for FACS staining and then for CyTOF staining is saturation of the epitope you stained for FACS. This can be solved in two ways by; finding antibody recognising a different epitope of the same marker or using secondary antibody against fluorescent dye that the FACS antibody was conjugated to.
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.
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.
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.
Answer No – any microscope slide will work as long as it is made of glass.
Answer Standard 4-7 μm thickness is ideal.
Answer Yes, both FFPE tissue and frozen tissue can be used successfully with the Hyperion.
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.
Answer There are a few ways in which you can check that your antibodies are suitable for use with the Hyperion. Firstly, antibodies can be tested for use in standard immunohistochemistry using a secondary antibody for detection. This is useful for determining whether your antibody is still functional after metal conjugation, or to get an idea of whether a previously metal-conjugated antibody may work for use in FFPE tissue. However, this method will not tell you if your metal conjugation was successful and will not give you the optimal dilution for detection with the Hyperion. A second method for determining whether antibodies are still functional after conjugation is to use coated beads and run these through the Helios. This will tell you that your antibody is capable of binding and that it is successfully conjugated with metal. However, this will give no information about which dilution to stain your samples at and will also not tell you whether the antibody is able to detect your specific target in tissue. The best way to determine whether your antibody works is to test it in your tissue of interest at multiple dilutions. If you are not sure if one of your targets is expressed in your tissue of interest, we also recommend that you validate this antibody on positive control tissue.