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Overview


If you're interested in what's involved in planning a mass cytometry project or are planning a project for yourself, then you've come to the right place. Here, we'll spell out the may things you should think about when planning your project. A little planning will let you reap the highest rewards.

A mass cytometry project workflow consists of:
  1. Define your scientific question
  2. Panel Design
  3. Reagent Acquisition
  4. Reagent Validation
  5. Sample Staining (and Optimization)
  6. Sample Acquisition
  7. Data Analysis

Your Scientific Question


It's is very important that you have a clear scientific question in mind. This question defines the markers we need to analyze, which defines your panel. 40-50 parameters are a lot in cytometry terms but relatively small compared to other -omics platforms. Without a clear question in mind it's very easy to acquire precious samples and realize we haven't measured everything we needed. We need to use each and every marker to drill down on your specific question.

Panel Design


Panel design is the process of pairing up your markers of interest with the instrument's channels. We do this so that when we acquire your samples, we attain the highest resolution and lowest background possible.

When designing your panel:
  • Assign the most important markers to the most sensitive channels
  • Assign highly expressed markers to the least sensitive channels
  • Ensure markers expressed on the same cell are not neighbours

Panel Design

Panel Design is analogous to planning the seating chart for a dinner party or wedding. We want our most important guests at the head of the table where they can be seen and heard easily. We want our brash loud uncles further down the table out of the way of quieter guests (-people will be able to hear him anyway no matter where we seat him). We also want to avoid clashes (maybe we seat the in-laws apart).

Design a big panel at the start!

We can stretch the analogy a little further to make one more point - In the same way we wouldn't plan a seating chart with gaps at the head of the table (just in case someone shows up), we shouldn't leave gaps in our panel design. It is much better to design a large panel now and not use all of it for initial studies than to design a small panel now and hope to expand it further down the line.

Reagent Selection

PRE-CONJUGATED ANTIBODIES

Antibodies already conjugated to lanthanide metals are primarily available from Fluidigm. They have an extensive catalogue of marker, especially if you are working in:

  • Immunology
  • Immuno-Oncology
  • Developmental Biology

CONJUGATING ANTIBODIES

If you're working in other fields you'll have to source some of your antibodies from other vendors (your usual antibody vendors will be fine). Purchasing antibodies for conjugation they should be:

  • Carrier-Free
  • IgG
  • Monoclonal (preferred)
  • 100ug

We can conjugate these antibodies to any lanthanide for you.

Reagent Validation

At least a few of your markers may never have been used in mass cytometry before (or at least not on the cells and tissues that you are using). It's very important to spend some time validating your antibodies for specificity and titrating ALL of your antibodies


Sample Preparation

As important as your panel design is, getting your sample preparation right. Optimizing your sample preparation helps immeasurably downstream steps like staining and acquisition. Your aim is to:

  • Generate a single cell suspension
  • Reduce Debris and Cell Death
  • Reduce Environmental Contamination

Sample Staining

Once you've mastered and optimized sample preparation you can stain your samples. If you're unfamiliar with cytometry techniques, a typical staining workflow consists of:


Single cell suspension → Surface Staining → Fixation → Permeabilization → Intracellular Staining


In mass cytometry fixation is always done with 1.6%-4% PFA. All samples must be fixed in mass cytometry experiments. Permeabilaization reagents vary depending on the targets being stained and can range from Methanol to Saponin to Tween-20 or Triton-X.

Sample Acquisition

At OHRI, we acquire samples on a 3rd-generation Helios instrument with top-of-the-line sensitivity and the highest mass range available. If you're more used to flow cytometry there are differences that you should be aware of:

  • 500 events / second acquisition rate
  • Samples are acquired in ddH2O

Data Analysis


For mass cytometry we rarely use manual gating techniques. Most often we use high parameter workflows consisting of:

  • Dimensionality Reduction
    • e.g. tSNE, hSNE, UMAP
  • Clustering
    • e.g. Phenograph, FlowSOM, SPADE
  • Trajectory Analysis
    • e.g. Pseudotime

We're here to help

The information provided here is a very brief introduction to many of the things you should be thinking about when planning a mass cytometry project. We appreciate that these are complex topics. If you have any questions about any aspect, let us know. We're here to help! Email us at dcarragher@ohri.ca with any queries