How to prepare samples for the Helios Mass Cytometer

So you're ready to prepare some samples for acquisition on the Helios. There's a few points that you should watch out for with mass cytometry samples. Follow the tips here to make sure that your samples remain uncontaminated and run well on the instrument.

The 3 goals of Sample Preparation

How big should your samples be?

If you're just starting out we recommend that you start with samples in the 2-3 million cell range. Sample preparation and staining can be quite harsh on cells, especially when you're learning. Cells are normally washed (centrifuged) multiple times, fixed and permeabilized with either detergents or alcohols. All of these treatments can lead to significant cell loss, so while you're learning it's better to use a larger sample to optimize your protocols. Don't worry. We can acquire samples as small as 25-50,000 cells but while you're optimizing your protocol, it's better to go big!

LEAD CONTAMINATION

Lead is used in glass manufacture. It can leech into any cells, buffers or media that have been stored in glass bottles and remains a contaminant in those cells for a long period of time. It's important to avoid any glassware in your sample preparation protocols.

BARIUM CONTAMINATION

Barium is found primarily found in dishwashing powders. It contaminates any glass- or plastic-ware washed in the glass-wash. It's vital to avoid using any glass-washed labware in your sample preparation. Ideally, you should use dispoasble plastics for every step of your assay.

IODINE CONTAMINATION

Iodine-containig compounds are an important component of centrifuge density gradients. It's crucial when using these gradients to be very careful when extracting the cell layer after centrifugation. It's better to leave some cells behind than take up too much of the media with your cells.

Single Cell Suspension

Mass cytometry analyses single cells. Cell doublets and debris confound analyses. A typical staining protocol for mass cytometry is: Single cell suspension → Surface Staining → Fixation → Permeabilization → Intracellular Staining. Generating a single cell suspension is the very first step in this process. If you make a good single cell suspension, it sets up your workflow. If you make a poor single cell suspension, it doesn't matter how well you perform subsequent steps, you'll have problems.

Difficult Tissues and Samples

If your tissues or cells of interest are particularly difficult to process to a debris-free, single cell solution there are options. There are proprietary kits that can be used remove both dead cells and debris. We've had good luck with the Miltenyi kits for removing either dead cells or debris but others are available.

Reduce Cell Death and Debris

Wash

Whether you're working with a cell line or solid organ it's crucial to wash your samples with PBS to remove debris, dead cells and blood.

Reduce Cell Death and Debris

Mechancial Disaggregation

Mechanical disaggregation, with a cell scraper for cell lines or with surgical tools for solid organs can be sufficient to generate a single suspension (e.g. thymus and spleen). Otherwise, mechanical disaggregation is useful to increase the surface area of the tissue for enzyme disaggregation.

Reduce Cell Death and Debris

Enzymatic Disaggregation

For solid tissues you'll often need to use enzymes like trypsin and collagenase to dissagregate them to a single cell suspension. If not carefully optimized this step wil be a major contributor to debris and dead cell accumulation

• Remember to use DNAse and EDTA to retard clumping due to dead cells
• Titrate your proteases and collagenases
• Check optimum temperature
• Check optimum incubation time
• Remember there are milder alternatives e.g. accutase.

Reduce Cell Death and Debris

Red Blood Cell Lysis

Erythrocytes significantly contribute to cell clumping, which causes loss of sample and adds to the debris burden of the sample. They are easy, quick and cheap to remove from your samples using ACK or any proprietary red blood cell lysis buffer.

Filtration

Filtering is a final step that can remove undigested tissue, larger aggregates and clumps. Use a 40-70um filter at the end of your sample preparation.