1. Plate-based Methods

This was the first method to appear and be published. Cell isolation is done by placing one cell in one well. This can be done manually under the microscope or in an automated way using an automatic cell dispenser, or FACS. If using FACS, we can even do a simple multiomic approach. Prior to FACS, we stain our cells with one or more antibodies against the protein (s) of interest. After that, we will do index sorting. Basically, we will save the characteristics of each cell and the plate well where it is localized. After that, we will be able to correlate this information with the genomic or transcriptomics information of each cell. Example: Cell 1A is expressing protein B and has five apoptotic genes up-regulated.

The essential material for this method are plates. They can be 96- or 384-well plates. Some commercial options available based on this type of method are: REPLI-g Advanced DNA Single Cell Kit (Qiagen), SMART-Seq Single Cell Kit (Takara Bio), ResolveDNA or ResolveOME (Bioscryb), or Evercode Whole Trancriptome (Parse Biosciences). With the exception of Parse Biosciences technology, plate-based approaches are low-throughput assays. So, if you have between a dozen and a hundred cells, you can use these approaches.

 2. Micro, Nano, or Pico-based Methods

Similar to the plate-based methods, here we also use wells to individualize cells. But in this case, the wells are very tiny. Depending on the commercial brand, we can have micro, nano, or pico-wells. These wells make part of a chip. After we load the cell suspension on the chip, the cells will fall by gravity into the wells. To be sure that we have one cell per well, we need to use the recommended cell concentration. This type of approach is a high-throughput assay, giving us the option of analyzing large numbers of cells. Some commercial options are: HIVE scRNAseq (Honeycomb Biotechnologies), GEXScope single cell RNA kits (Singleron), the ICELL8 system (Takara Bio) and BD Rhapsody system (BD Biosciences).

This type of method is ideal in cases where we want to study sensitive cells, like granulocytes or neutrophils. The main reason is that the cells enter the wells by gravity, not due to forces that can make them "explode", what makes this type of method very gentle with the cells. Some commercial options even allow us to load our cells into the chip, store them, and perform the experiment days later. This can be crucial for projects where the sample collection site is different from the laboratory site or when the samples cannot be processed on the same day of collection.

 3. Droplet-based Methods

These are the most widely used and known single-cell methods. Some commercial options available are those from 10X Genomics (Chromium Controllers), Mission Bio (Tapestri), Dolomite Bio (Nadia), or MGI (DNBelab C Series).

To use this type of assays, we will need a machine that will be able to individualize one cell inside an oil droplet. Just for you to have an idea of how it works, let's imagine a thin horizontal capillary or channel where we will load our cells that are resuspended in a water-based mixture formed by all the constituents needed for our downstream application (e.g., DNTP, enzyme, etc.). In the middle of the horizontal channel, there will be a vertical channel crossing it. In this one, there will be a flow of unique labels that are small beads. One bead will be attached to one cell. After the cell is labelled with the unique bead, it will continue in the horizontal channel flow until it enters a pool filled of oil. When this happens, it will be formed automatically an oil droplet around the cell, being this the way of isolating it from the others. These droplets resemble kitchen droplets when we mix water and oil. Each droplet will work as a PCR reaction tube.

The droplet-based methods are high-throughput assays and they can be used in most part of the research projects. Since this type of method is the most developed, we will find more applications (e.g. genomics, transcriptomics, epigenomics, multiomics...).

Clarification: Sometimes people refer to the droplet-based methods as microfluidics. It is true because the machines are based on a microfluidic system. However, microfluidics is also the base of the chips (micro, nano, or pico-well-based methods). So, microfluidics refers to systems that can process small quantities of fluids by using tiny channels with dimensions at the micro-scale.

Now that you know the three main different types of single-cell methods, you can choose better the technology for you project. I hope you find this article useful.

Have a lovely day.

Cheers,

Cátia