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What is Fluorescence Activated Cell Sorting?

Fluorescence-activated cell sorting (FACS cell sorting) is a powerful tool used in both basic and clinical research that enables individual cells to be separated from a heterogeneous sample for downstream analysis or therapeutic applications. FACS sorting can be used to separate live cell populations into subpopulations based upon the fluorochrome associated with a detection antibody.

Typically, a FACS technician can sort 2-4 distinct bulk populations of cells from a heterogeneous cell mixture, or sort single-cells into 96 or 384 well plates for single-cell genomic, epigenetic, proteomic, glycomic, lipidomic or metabolomic studies.

Cell Sorting

FACS cell sorting is a specialized form of flow cytometry and uses many of the same principals of cell preparation and labeling as traditional cytometry. In fact, a cell sorter works in a similar way to a flow cytometer with a single-cell suspension of fluorescently labeled cells passing through a fluidic system and interrogated by lasers that excite the fluorescent molecules used to label the different cell types. However, uniquely to cell sorters, electrostatic drop deflection, (the same principal used in ink-drop printers), is employed to target specific cell types for collection.

This is achieved by utilizing a vibration mechanism to create break points in the fluidics stream that result in the generation of distinct droplets. By fine-tuning the frequency of the vibrations, the frequency of the break points can be optimized to generate droplets containing single cells. Charge (hi/low positive or negative) is applied to droplets containing cells of interest, enabling them to be diverted into collection tubes using electrostatically charged deflection plates.

This enables relatively pure cell populations to be collected. Typically, 2 to 4 different cell populations can be sorted simultaneously from a single heterogeneous population. Populations can be sorted based on a variety of characteristics – essentially, if you can differentiate a cell, you can sort it.

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Types of Differentiating Characteristics that Enable Cell Sorting

  • Surface Binding Properties Icon

    Surface Binding Properties

  • Number of Bound Fluorochromes Icon

    Number of Bound Fluorochromes

  • Membrane Composition (BODIPY or Nile Red binding) Icon

    Membrane Composition (BODIPY or Nile Red binding)

  • Scatter Properties (SSC, FSC)- Size and Complexity of the Cell or Particle Icon

    Scatter Properties (SSC, FSC)- Size and Complexity of the Cell or Particle

  • Expression of fluorescent target protein e.g. Green Fluorescent Protein Icon

    Expression of fluorescent target protein e.g. Green Fluorescent Protein

  • Level of Fluorescence (note, this differentiates cell sorting from magnetic bead sorting) Icon

    Level of Fluorescence (note, this differentiates cell sorting from magnetic bead sorting)

Applications of Cell Sorting

  • Transplantation

    Cell line development-
    isolation of transfected/transduced cells

    Establishing cell clones-
    (protein engineering, binding, and enzymatic studies)

    Rare Cell Population Studies

  • Immune Cell Profiling

    Omic Studies-
    protein/DNA/ RNA; single-cell qPCR

    Subcellular Particle Analysis (microvesicles, exosomes, microbial isolation) enriching nuclei for single-cell epigenomics.

    Screen CRISPR libraries,
    Gene Editing Programs

The Process of Cell Sorting

  • Preparation and maintenance
    of a highly viable, single-cell suspension
  • Live/Dead and surface staining
    to label population of interest
  • Selection of sort nozzle selection,
    breakpoint frequency and sort speed optimization
  • Collect up to 4 separate populations
    of cells into optimized media or buffer
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Key Considerations Before Starting a Cell Sort:


What is the downstream assay?

This drives the main features of the sort: purity, yield, or speed of sorting. The higher the purity requirements, then the slower the sort and lower the yield. If speed is of the essence, then the final sorted population may be enriched in the target cells, but the purity may be compromised.

Sort Precision Mode Sorting Features
Yield High Yield, Low Purity- depending on flow rate
Purity High Purity, Low Yield- depending on flow rate
Single-Cell Highest Purity, lowest yield

What is the required number of sorted cells?

Plan for enough starting material to recover this target based on the % of these cells in the heterogeneous sample. Sort speeds using a 70µm nozzle are around 30,000 cells per second. For sorting rare cells such as TDC that represent < 0.1% of the cell population it may be possible to only recover 2.5x106 cells/day of sorting.

The efficiency and time involved in sorting rare cell populations can be improved by pre-enrichment of the sample prior to sorting using either depletion of common cell types or capture of target cell types using Ficoll™ gradients or magnetic bead selection. These approaches can greatly shorten the downstream sorting time required to sort the target population, as well as improve yields and purity.

Optimizing the Process of Cell Sorting

Selection of nozzle size is important. Typically, the size of the cells should be less than 1/3rd of the nozzle diameter. Below are some of the cell types routinely sorted at FlowMetric, long with the nozzle size that is employed.

Nozzle Diameter Characteristics of Cell Sort Types of Cells Sorted
70µm Nozzle Used for smallest cells, particles <10µm- fastest sort times E.coli, RBCs, Bone Marrow, Splenocytes, Lymphocytes
100µm Nozzle Used for the majority of cell types <20µm CHO, HEK, Stem Cells, Macrophages, Lymphoid, Cell Lines
130µm Nozzle Used for large, fragile cells- slowest sort times HeLa, Hybridoma, Neurons, Fibroblast, CHO, Embryonic Stem Cells
Nozzle Diameter

70µm Nozzle

Characteristics of Cell Sort


Used for smallest cells, particles <10µm- fastest sort times

 

Types of Cells Sorted


E.coli, RBCs, Bone Marrow, Splenocytes, Lymphocytes

 

100µm Nozzle

Characteristics of Cell Sort


Used for the majority of cell types <20µm

 

Types of Cells Sorted


CHO, HEK, Stem Cells, Macrophages, Lymphoid, Cell Lines

 

130µm Nozzle

Characteristics of Cell Sort


Used for large, fragile cells- slowest sort times

 

Types of Cells Sorted


HeLa, Hybridoma, Neurons, Fibroblast, CHO, Embryonic Stem Cells

 

Successful FACS cell sorting requires a well dispersed, single-cell suspension of high viability cells. When handling adherent cell lines, we recommended using soybean trypsin inhibitor in place of FBS to neutralize which helps to reduce cellular auto-fluorescence. Cell clumping is a significant issue for cell sorting since it can result in clogs and impact the purity and yield of sorting. Sticky cell suspensions should therefore be treated with DNase+ 5mM MgCl2 to remove free-floating DNA and be sorted at a lower concentration of cells. Harvested cell suspensions should be maintained on ice in Ca++/Mg++-free PBS to minimize aggregation.

Cell filtration prior to flow cytometry analysis is always recommended but is essential for FACS cell sorting protocols to ensure no clumps are present, and the use of a live/dead stain such as PI, DAPI, or 7-AAD is highly recommended. Avoid buffers containing Sodium Azide since this can be toxic to cells and detrimental to cell viability. It is important to note that the high pressures involved in cell sorting can cause the sort buffer to become more basic; in this event, the addition of 25mM HEPES should help maintain a pH of 7-8.

Throughout the process of cell sorting, it is important that the sample be…

kept on ice to help reduce cell degradation

sorted into an appropriate media, conditioned media, serum, or buffer depending upon the downstream applications for these cells

FlowMetric can provide guidance on all aspects of the cell preparation, sorting, and collection methodology depending on your specific program needs.

 

As a rule of thumb, the stickier the cell preparation, the lower the cell density during the sort; for example, adherent cells are typically sorted at a concentration of 5-10x106 cells per mL using a 100µm diameter nozzle, whereas lymphocytes can be sorted at concentrations of 10-20x106 cells per mL using the 70µm diameter nozzle.

Since intracellular staining involves cell permeabilization and fixation, it is not possible to sort live cells based on an intracellular marker. An optimal sort buffer is low in protein to minimize autofluorescence, examples of this type of buffer include PBS with 1% dialyzed FBS, or commercially available preparations.

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Optimization of the Gating Strategy to Sort Your Target Cells Most Efficiently

Gating strategies for flow cytometry and cell sorting are very similar. The use of a live/dead stain such as 7-AAD is always recommended, and FSC versus viability dye signal is a simple way to exclude dead cells. It is also critical to remove doublets that could potentially impact the purity of the sorted samples. This is typically achieved using pulsed geometry gating such as FSC-H versus FSC-A. Compensation controls and fluorescence minus one (FMOs) controls should also be considered in order to provide confidence in the accuracy and robustness of the cell sort.

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A. PBMCs from immunized camelids were stained with fixable viability dye and fluorescently conjugated human and cynomolgus macaque antigen. The cells were gated on the live population, followed by a singlet lymphocyte gate. Antigen-specific B-cells were then sorted. The B-cells specific to the cynomolgus protein were identified in gate P4; B-cells specific to the human protein were identified in gate P5; and cross-reactive to proteins from both species were identified in gate P6. The three separate B-cell populations were then sorted.


Example of Cell Sorting of Camelid B-cells Example of Cell Sorting of Camelid B-cells chart

B. PBMCs from immunized camelids were stained with fixable viability dye and fluorescently conjugated SARS-CoV-2 antigens: M2 protein and S/M fusion antigen. The cells were gated on the live population, followed by a singlet lymphocyte gate. Antigen-specific B-cells were then sorted. The B-cells specific to the SARS-CoV-2 S/M fusion antigen were identified in gate P4; B-cells specific to the SARS-CoV-2 M2 protein were identified in gate P6. The two separate B-cell populations were then sorted.

Example of Cell Sorting of Camelid B-cells chart

How FlowMetric Supports Cell Sorting Projects

FlowMetric can provide FACS cell sorting services according to your specific program needs. We provide aseptic sorting or non-aseptic sorting within BSL-2 safety cabinets. Our BD FACS Aria III instruments support bulk 4-way population sorting based on up to 16 parameters, or single-cell sorting into 96 or 384 well plates.

FACS cell sorting is a powerful tool that provides unmatched utility in both translational and clinical research. Optimal cell sorting requires considerable planning and technical optimization. Working with FlowMetric’s cell sorting experts in the design and execution of sorting runs will help to ensure your cell sorting yields and purities are achieved. Consider the many ways sorting can enhance your research projects.

The FlowMetric Advantage

The FlowMetric team are industry experts in the development and validation of high-complexity flow cytometry panels. Our team includes scientific and quality management experts who specialize in efficient panel design and the adoption of fit-for-purpose validation test scripts. All performed with a commitment to quality to ensure that every aspect of the method, analytics, and reporting meet the regulatory requirements for your assay’s intended use.

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