Frequently Asked Questions
Find answers to your questions about the Spatial Molecular Profiling Shared Resource.
Mass cytometry combines the advantages of single-cell, high-speed analysis common to conventional flow cytometry with the ability to resolve more than 100 metal probes with minimal signal overlap common to atomic mass spectroscopy, thereby providing researchers with an unparalleled ability to phenotypically and functionally profile cells from normal and diseased states.
Helios™ is the latest generation of the mass cytometer system. It is designed to analyze metal-tagged antibodies in cells suspended in solution.
The Hyperion™ Imaging System includes a separate module that can be connected to Helios to analyze metal-tagged antibodies in tissue. It uses laser ablation and can relate the metal tags to their spatial position on a slice of tissue.
- Pre-project consulting on research project objectives, resource planning, antibody panel design and data analysis tools
- Training on antibody conjugation and staining to core users
- Data acquisition
- Antibody conjugation and sample staining by IMC core personnel (planned for July 2021)
- IMC data processing (denoising, pixel classification training and cell segmentation, clustering and spatial analysis) (planned for July 2021)
All core lab requests are made through iLabs.
Yes, we can provide training, protocols and references for published studies.
Project planning > Antibody panel design > Antibody validation and titration > Staining of samples > Data acquisition > Data QC and processing > Data analysis.
The core lab will provide basic panel templates that include empty channels so users can modify the panel to their project.
A panel builder web app from Fluidigm can be found at: https://dvssciences.com/paneldesigner/resources.
Yes, the SMPSR core has had experience with both human and mouse cells and tissues.
All users must validate their own antibodies on the cells or tissues they are using for their studies. Antibodies should be demonstrated to work first by flow, IHC or IF, then they can be finally validated by mass cytometry before starting the study. Control tissues can be provided by the Biobank histology core lab for SMPSR.
No. Users are not allowed to operate the instrument. Only core personnel can use the instrument.
Antibodies are tagged with rare-earth metal isotopes from the Lanthanide series. Suspension cell panels can accommodate 48 markers, while SMPSR panels can contain up to 40 markers at this time.
The instrument has a range of 75-209 atomic mass units (AMU), these metals do not occur naturally in living organisms so there is no possibility of contamination from the biological sample. Channels are limited to those metals that can be obtained at a minimum of 95% monoisotopic purity, although ≥98% purity is preferred to reduce the possibility of spillover into neighboring channels.
This spillover experiment is described here, and the software used to analyze and apply this information can be found here.
Imaging Mass Cytometry (IMC) is a mass-spectrometry-based tissue imaging platform that uses high-resolution laser ablation followed by transfer of the ablated materials to the mass cytometer for time-of-flight detection of the metal ions to generate high-dimensional image data that enables comprehensive analyses of target protein expressions, cell types, their functional states and spatial interaction.
Immunofluorescence (IF) is based on fluorophores that are excited by one wavelength and emit at a longer wavelength. Multiplex studies using IF are challenged by the broad emission spectra of most organic fluorochromes, leading to overlap or crosstalk between channels, which limit the number of target proteins that can be measured simultaneously.
Using metal-conjugated antibodies instead of fluorochromes, IMC allows simultaneous detection of up to 40 protein targets in a single round of staining while eliminating the common challenges of conventional IF such as signal fading, spectral overlap and autofluorescence.
With 1 µm laser beam size, IMC enables visualization and analyses of tissues at subcellular spatial resolution. (a typical immune cell is 5-7 microns in diameter).
We can work with any tissues from any organism that can be easily sectioned to 4-5 µm and have known antibodies that can be used for cellular markers. IMC is compatible with both OCT embedded frozen sections and FFPE sections.
The core works primarily with FFPE-derived tissue sections because they are clinical pathology samples. For optimal IMC assay performance, FFPE tissues should be appropriately fixed, with cold ischemia time of less than 1 hour.
Using frozen sections may allow for more choices of antibodies because flow cytometry antibodies can often be used.
Yes. However, we are currently not accepting samples fixed with B5 due to mercury content. Depending on the method of decalcification, certain antibodies may not function the same, so all antibody panels should be validated on the appropriate control tissues before being used on experimental samples.
Any type of charged glass slides of 25 x 75 mm used for IHC is compatible with IMC.
The tissue sections should be cut in the 4-5 µm range for optimal single-cell resolution, although sections up to 10 µm have been routinely run without a problem, single-cell resolution might suffer.
For optimal data quality, the tissue sections should be cut fresh, or at most within a week from the date of IMC staining. We have run tissues that are up to 6 months old after cutting although antibody performance can't be guaranteed.
Specific areas of tissues on the slides that capture the essential structural and functional heterogeneity of the diseases under investigation. These tissues areas are called "Regions of Interest" or ROIs and will be ablated using the to generate IMC data. ROIs should be identified on either an H&E or IHC slide often by a pathologist.
A typical ROI size for IMC data acquisition is 1 mm2. However, larger or smaller areas can be selected If needed. We suggest acquiring the smallest ROI needed to address the scientific question in order to conserve time and resources.
With 200 Hz laser frequency (200 laser shots per second), it takes approximately 1.5-2 hours to ablate an ROI of 1 mm2.
If it's a large resection specimen, multiple ROIs are usually ablated to capture the heterogeneity of the tumor and immune microenvironment.
The Hyperion Tissue Imager can only hold one slide at a time. If the project involves multiple tissue slides, the lead time to complete data acquisition will be longer given the time it takes to exchange slide and recalibrate the Hyperion. Ideally, a pathologist would preselect areas (cores) of the tissues that are representative of the tumors and their microenvironment then construct a tissue microarray (TMA) from those tissue cores. Using a TMA for IMC experiment not only eliminates the slide exchange and equipment recalibration time, but it also saves the cost of antibodies needed for staining.
Typical TMA core size ranges from 1-2 mm. Depending on the size of the TMA cores, there can be one or two ROI(s) ablated per core. For example, a TMA of 20 one-millimeter cores will have 20 ROIs selected for ablation. Replicates core from one tumor should be placed on separate TMAs to allow for identification of batch effects during analysis.
The top and bottom 4.5 mm areas (grey and yellow zones) of the slide are not accessible for ablation by the Hyperion. When cutting a TMA section for IMC experiment, it is recommended to place the tissue section at the center of the slide (purple zone).
To give an idea of how big a TMA section can be to fit perfectly on a slide for IMC studies, the purple accessible area on a TMA slide can fit up to five horizontal rows and 20 vertical columns of 1.5 mm cores.
Given a slide containing tissue area of approximately 10 mm2, the average cost of a validated, pre-conjugated antibody in Fluidigm's catalog is typically $50 per experiment but can range between $25-$75. So, if the panel contains 30 antibodies, the estimated cost of antibodies for one round of staining is $1,500/per slide. Actual costs would depend on size of tissue being staining, whether pre-conjugated antibodies were purchased from Fluidigm or customer conjugated antibodies were used, and the final titer of the antibody used in the experiment.
Sample prep is very similar to what is done for traditional flow cytometry, meaning that cell suspensions are stained in tubes with a panel of tagged antibodies directed against an array of cellular protein targets. It is important to use metal-free sample processing buffers (see our catalog). The final staining step includes a nucleic acid intercalator that is used to identify nucleated events.
Yes. The standard protocols for staining intracellular targets is similar to flow cytometry and involves fixing the cells followed by permeabilization. As with flow, some cellular epitopes are altered by fixation and/or permeabilization, which can affect antibody binding. Optimization of antibody clone and/or fix/permeabilization conditions will usually enable detection of the problematic marker.
Yes, cell cycle stages can be determined using 127IdU and metal-tagged antibodies to pRb, cyclin B1, and pH3. The method is described in detail in Behbehani et al. 2012. Briefly, G0 cells are negative for all four markers, G1 cells express only pRb, S phase cells incorporate 127IdU during DNA replication, G2 cells express cyclin B1 and mitotic cells uniquely express pH3.
PBMCs can be stored for up to two weeks in PBS plus 2% formaldehyde at 4 degrees Celsius. Metal-labeled antibodies are not light sensitive. Researchers should test their cells with the panels to determine maximum storage time.
With optimization and barcoding, an experiment with 40 conjugated antibodies can cost under $70 per sample, including acquisition time. This price is assuming all antibodies are conjugated in the lab. Pre-conjugated antibodies from Fluidigm generally cost $421 for 100 tests.
Most antibodies validated for traditional flow cytometry will work as metal conjugates in mass cytometry.
No, at this time users must purchase their own antibodies. Fluidigm has many metal-labeled antibodies that are validated for both suspension cells and IMC. Purified, carrier protein-free antibodies can be purchased from various vendors and conjugated to metal tags using conjugation kits.
Antibodies and metal conjugation kits can be purchased from Fluidigm. A limited catalog of antibodies that can be purchased a la carte is available through the core for pilot projects.
Yes. The core can provide the core users with training session as well as protocols for antibody conjugation and staining.
Antibody conjugation kits use a simple chemistry, and the process takes a few hours. Most of that time is consumed by incubations and washes. The labeling protocol works for all metals.
Conjugation kits work for most IgG antibodies. We have had less success with IgM, though it has worked for some. This is likely because the structure of IgM hinders access to SH groups necessary for covalent binding to the metal-labeled polymer. We will be able to share information about clones that have worked and not worked.
Each 4-reaction antibody labeling kit generally costs $559 and contains sufficient reagents to conjugate 4 antibodies in 100 ug amounts. Included in the kit are 4 tubes of polymer with lanthanide (Ln) metal isotopes and 4 bottles of buffers.
Have Questions or Need Help?
Contact us if you have questions or would like to learn more about the Spatial Molecular Profiling Shared Resource at Cedars-Sinai.
Spatial Molecular Profiling Shared Resource
127 S San Vicente Blvd.
AHSP A8700
Los Angeles, CA 90048